Compositions and their uses directed to il-4r alpha

ABSTRACT

Disclosed herein are compounds, compositions and methods for modulating the expression of IL-4R alpha in a cell, tissue or animal. Also provided are methods of target validation. Also provided are uses of disclosed compounds and compositions in the manufacture of a medicament for treatment of diseases and disorders related to expression of IL 4R-α, airway hyperresponsiveness, and/or pulmonary inflammation.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationSer. Nos. 60/656,760, filed Feb. 25, 2005; 60/688,897, filed Jun. 9,2005; 60/700,656, filed Jul. 19, 2005; and 60/709,404 filed Aug. 18,2005; all of which are incorporated herein by reference in theirentirety.

INCORPORATION OF SEQUENCE LISTING

A copy of the sequence listing in both a paper and a computer-readableform is provided herewith and hereby incorporated by reference. Thecomputer readable form is provided on 3.5″ diskette containing the filenamed RTS0792WOSEQ.txt

BACKGROUND OF TH E INVENTION

Allergic rhinitis and asthma are widespread conditions with complex andmultifactorial etiologies. The severity of the conditions vary widelybetween individuals, and within individuals, dependent on factors suchas genetics, environmental conditions, and cumulative respiratorypathology associated with duration and severity of disease. Bothdiseases are a result of immune system hyperresponsiveness to innocuousenvironmental antigens, with asthma typically including an atopic (i.e.,allergic) component.

In asthma, the pathology manifests as inflammation, mucusoverproduction, and reversible airway obstruction which may result inscarring and remodeling of the airways. Mild asthma is relatively wellcontrolled with current therapeutic interventions includingbeta-agonists and low dose inhaled corticosteroids or cromolyn. However,moderate and severe asthma are less well controlled, and require dailytreatment with more than one long-term control medication to achieveconsistent control of asthma symptoms and normal lung function. Withmoderate asthma, doses of inhaled corticosteroids are increased relativeto those given to mild asthmatics, and/or supplemented with long actingbeta-agonists (LABA) (e.g., salmeterol) or leukotriene inhibitors (e.g.,montelukast, zafirlukast). Although LABA can decrease dependence oncorticosteroids, they are not as effective for total asthma control ascorticosteroids (e.g., reduction of episodes, emergency room visits)(Lazarus et al., JAMA. 2001. 285: 2583-2593; Lemanske et al., JAMA.2001. 285: 2594-2603). With severe asthma, doses of inhaledcorticosteroids are increased, and supplemented with both LABA and oralcorticosteroids. Severe asthmatics often suffer from chronic symptoms,including night time symptoms; limitations on activities; and the needfor emergency room visits. Additionally, chronic corticosteroid therapyat any level has a number of unwanted side effects, especially inchildren (e.g., damage to bones resulting in decreased growth.

Allergic rhinitis is inflammation of the nasal passages, and istypically associated with watery nasal discharge, sneezing, congestionand itching of the nose and eyes. It is frequently caused by exposure toirritants, particularly allergens. Allergic rhinitis affects about 20%of the American population and ranks as one of the most common illnessesin the US. Most suffer from seasonal symptoms due to exposure toallergens, such as pollen, that are produced during the natural plantgrowth season(s). A smaller proportion of sufferers have chronicallergies due to allergens that are produced throughout the year such ashouse dust mites or animal dander. A number of over the countertreatments are available for the treatment of allergic rhinitisincluding oral and nasal antihistamines, and decongestants.Antihistamines are utilized to block itching and sneezing and many ofthese drugs are associated with side effects such as sedation andperformance impairment at high doses. Decongestants frequently causeinsomnia, tremor, tachycardia, and hypertension. Nasal formulations,when taken improperly or terminated rapidly, can cause reboundcongestion. Anticholinergics and montelukast have substantially fewerside effects, but they also have limited efficacy. Similarly,prescription medications are not free of side effects. Nasalcorticosteroids can be used for prophylaxis or suppression of symptoms;however, compliance is variable due to side effects including poor tasteand nasal irritation and bleeding. Allergen immunotherapy is expensiveand time consuming and carries a low risk of anaphylaxis.

Persistent nasal inflammation can result in the development of nasalpolyps. Nasal polyps are present in about 4.2% of patients with chronicrhinitis and asthma (4.4% of men and 3.8% of women) (Grigores et al.,Allergy Asthma Proc. 2002, 23:169-174). The presence of polyps isincreased with age in both sexes and in patients with cystic fibrosisand aspirin-hypersensitivity triad. Nasal polyposis results from chronicinflammation of the nasal and sinus mucous membranes. Chronicinflammation causes a reactive hyperplasia of the intranasal mucosalmembrane, which results in the formation of polyps. The precisemechanism of polyp formation is incompletely understood. Nasal polypsare associated with nasal airway obstruction, postnasal drainage, dullheadaches, snoring, anosmia, and rhinorrhea. Medical therapies includetreatment for underlying chronic allergic rhinitis using antihistaminesand topical nasal steroid sprays. For severe nasal polyposis causingsevere nasal obstruction, treatment with short-term steroids may bebeneficial. Topical use of cromolyn spray has also been found to behelpful to some patients in reducing the severity and size of the nasalpolyps. Oral corticosteroids are the most effective medication for theshort-term treatment of nasal polyps, and oral corticosteroids have thebest effectiveness in shrinking inflammatory polyps. Intranasal steroidsprays may reduce or retard the growth of small nasal polyps, but theyare relatively ineffective in massive nasal polyposis. Although nasalpolyps can be treated pharmacologically, many of the therapeutics haveundesirable side effects. Moreover, polyps tend to be recurrent,eventually requiring surgical intervention. Compositions and methods toinhibit post-surgical recurrence of nasal polyps are not presentlyavailable.

Other diseases characterized by similar inflammatory pathways include,but are not limited to, chronic bronchitis, pulmonary fibrosis,emphysema, chronic obstructive pulmonary disease (COPD), and pediatricasthma.

Interleukin Receptor 4-Alpha and Inflammatory Signaling Pathways

It is generally acknowledged that allergy and asthma are a result of thedysregulation of the Th2 cytokine response. The presence of CD4+ T cellsproducing interleukin 4 (IL 4), IL 5 and IL 13 cytokines inbronchoalveolar lavage fluid and in airway epithelial biopsies ofasthmatics has been clearly documented. Neutralization of IL 5 resultsin a decrease in eosinophilia in man, in the absence of a reduction inairway hyperresponsiveness (AHR). IL 4 and IL 13 have been implicated inmultiple pathological processes that underlie asthma and allergy,including Th2 lymphocyte differentiation, induction of immunoglobulin E(IgE) production via regulation of the Ig isotype switch to the epsilonheavy chain in B lymphocytes, upregulation of IgE receptors and vascularassociated adhesion molecule-1 (VCAM-1) expression, promotion ofeosinophil transmigration in the lung, and mucus hypersecretion. IL 13mediates the development of airway hyperresponsiveness (AHR) tocholinergic stimuli, lung remodeling, and promotion of the secretoryphenotype of the inflamed airway epithelium. These observations makecomponents of the Th2 cytokine pathway, particularly IL 4 and IL 13,potential targets for therapeutic intervention for asthma, allergy, andother forms of airway inflammation and/or hyperresponsiveness.

The IL 4 and IL 13 receptors share a common signaling chain, IL 4receptor alpha (IL 4R-α). IL 4R-α pairs with the common gamma chain oncells of hematopoietic origin to form a type I IL 4R. This receptorbinds exclusively IL 4. IL 4 and IL 13 also signal through a secondreceptor. The receptor is composed of IL 4R-α and IL 13R-α1 (type II IL4R). IL 13R-α1 is present on both hematopoietic and non-hematopoieticcells. Formation of the IL 4R-α and IL 13R-α1 heterodimer results in ashift in affinity of IL 13R-α1 from a low affinity receptor, to a highaffinity receptor. The IL 13R-α is a monomeric, high affinity IL 13receptor that is thought to act as a decoy receptor to negativelyregulate IL 13 activity. Signaling through the type I and type II IL 4Rsactivates the Jak-Stat pathway; insulin-interleukin-4 receptor (14R)motif associated factors such as insulin receptor substrate family ofproteins; SH2 containing tyrosine phosphatases; and members of the Statfamily such as Stat 6. A number of genetic studies have demonstratedthat both IL 4R-α and Stat 6 are essential for allergen-inducedpulmonary inflammation and AHR in mice.

IL 4R-α, was cloned independently by two groups (Galizzi et al., Int.Immunol., 1990, 2, 669-675; and Idzerda et al., J. Exp. Med., 1990, 171,861-873). The human IL4 receptor gene was localized to 16p11.2-16p12.1by in situ hybridization, and the mouse homolog was localized to thedistal region of chromosome 7. The position on human chromosome 16suggests that the IL4 receptor may be a candidate for rearrangements.For example, 12; 16 translocations are often associated with myxoidliposarcomas (Pritchard et al., Genomics, 1991, 10, 801-806).

Inhibitors of IL 4 and IL 13 independently have producedanti-inflammatory effects in mouse pulmonary inflammation models or inclinical trials (Wills-Karp M et al. Science-282: 2258-2261, 1998;Grunig G et al. Science 282: 2261-2263, 1998; Borish L C et al., Am JRespir Crit. Care Med 160: 1816-1823, 1999; Kumar R K et al., Am JRespir Crit. Care Med 170: 1043-1048, 2004; Yang G et al., Cytokine 28;224-232, 2004) and are currently being pursued as novel therapeutics forallergy and asthma.

Antisense Oligonucleotides Aid Pulmonary Disease

Antisense oligonucleotides (ASOs) are being pursued as therapeutics forpulmonary inflammation, airway hyperresponsiveness, and/or asthma. Lungprovides an ideal tissue for aerosolized ASOs for several reasons (Nyceand Metzger, Nature, 1997: 385:721-725, incorporated herein byreference); the lung can be targeted non-invasively and specifically, ithas a large absorption surface; and is lined with surfactant that mayfacilitate distribution and uptake of ASOs. Delivery of ASOs to the lungby aerosol results in excellent distribution throughout the lung in bothmice and primates. Immunohistochemical staining of inhaled ASOs innormalized and inflamed mouse lung tissue shows heavy staining inalveolar macrophages, eosinophils, and epithelium, moderate staining inblood vessels endothelium, and weak staining in bronchiolar epithelium.ASO-mediated target reduction is observed in dendritic cells,macrophages, eosinophils, and epithelial cells after aerosoladministration. The estimated half life of a 2′-methoxyethoxy (2′-MOE)modified oligonucleotide delivered by aerosol administration to mouse ormonkey is about 4 to 7, or at least 7 days, respectively. Moreover, ASOshave relatively predictable toxicities and pharmacokinetics based onbackbone and nucleotide chemistry. Pulmonary administration of ASOsresults in minimal systemic exposure, potentially increasing the safetyof such compounds as compared to other classes of drugs.

Compositions and methods for formulation of ASOs and devices fordelivery to the lung and nose are well known. ASOs are soluble inaqueous solution and may be delivered using standard nebulizer devices(Nyce, Exp. Opin. Invest. Drugs, 1997, 6:1149-1156). Formulations andmethods for modulating the size of droplets using nebulizer devices totarget specific portions of the respiratory tract and lungs are wellknown to those skilled in the art. Oligonucleotides can be deliveredusing other devices such as dry powder inhalers or metered dose inhalerswhich can provide improved patient convenience as compared to nebulizerdevices, resulting in greater patient compliance.

Generally, the principle behind antisense technology is that anantisense compound hybridizes to a target nucleic acid and effects themodulation of gene expression activity, or function, such astranscription or translation. The modulation of gene expression can beachieved by, for example, target RNA degradation or occupancy-basedinhibition. An example of modulation of target RNA function bydegradation is RNase H-based degradation of the target RNA uponhybridization with a DNA-like antisense compound. Another example ofmodulation of gene expression by target degradation is RNA interference(RNAi) using small interfering RNAs (siRNAs). RNAi is a form ofantisense-mediated gene silencing involving the introduction of doublestranded (ds)RNA-like oligonucleotides leading to the sequence-specificreduction of targeted endogenous mrRNA levels. This sequence-specificitymakes antisense compounds extremely attractive as tools for targetvalidation and gene functionalization, as well as therapeutics toselectively modulate the expression of genes involved in diseases.

Antisense oligonucleotides targeted to a number of targets including,but not limited to p38 alpha MAP kinase (US Patent Publication No.20040171566, incorporated by reference); the CD28 receptor ligands B7-1and B7-2 (US Patent Publication 20040235164, incorporated by reference);intracellular adhesion molecule (ICAM) (WO 2004/108945, incorporated byreference); and adenosine A₁ receptor (Nyce and Metzger, Nature, 1997,385:721-725) have been tested for their ability to inhibit pulmonaryinflammation and airway hyperresponsiveness in mouse, rabbit, and/ormonkey models of asthma when delivered by inhalation. Various endpointswere analyzed in each case and a portion of the results are presentedherein. ASOs targeted to p38 alpha MAP kinase reduced eosinophilrecruitment, airway hyperresponsiveness (AHR), and mucus production intwo different mouse models. ASOs targeted to each B7.1 and B7.2decreased target expression and eosinophil recruitment. An ASO targetedto B7.2 also reduced AHR. ASOs targeted to ICAM-1 decreased AHR anddecreased neutrophil and eosinophil recruitment in mice. Treatment ofCynomolgus monkeys with an ASO targeted to ICAM-1 significantly reducedairway impedance (resistance) induced by methacholine challenge innaturally Ascaris allergen-sensitized monkeys. An ASO targeted toadenosine A₁ receptor reduced receptor density on airway smooth muscleand reduced AHR in an allergic rabbit model. These data demonstrate thatoligonucleotides are effectively delivered by inhalation to cells withinthe lungs of multiple species, including a non-human primate, and areeffective at reducing airway hyperresponsiveness and/or pulmonaryinflammation.

However, treatment with any ASO targeted to any inflammatory mediatorinvolved in pulmonary inflammation is not always effective at reducingAHR and/or pulmonary inflammation. ASOs targeted to Jun. N-terminalKinase (JNK-1) found to decrease target expression in vitro were testedin a mouse model of asthma. Treatment with each of two differentantisense oligonucleotides targeted to JNK-1 were not effective atreducing methacholine induced AHR, eosinophil recruitment, or mucusproduction at any of the ASO doses tested.

A number of ASOs and siRNAs designed to target IL 4R-α have beenreported for use as research or diagnostic tools, or as pharmaceuticalsfor the treatment of respiratory disease. US Patent ApplicationUS20030104410 teaches an array of nucleic acid probes useful as researchtools to identify or detect gene sequences. Allelic variations in the IL4R-α gene have been identified that increase receptor signaling (Hersheyet al., NEJM, 1997, 337:1720-1725; Rosa-Rosa et al., J. Allergy Clin.Immunol. 1999, 104:1008-1014; Kruse et al., Immunol., 1999, 96,365-371). PCT patent application WO 2000034789 teaches oligonucleotidesfor use in diagnostic testing to detect these allelic variations. Patentapplications WO 2002085309, WO 2004011613 and US 20040049022 teach ASOstargeted to a series of genes potentially relevant to respiratorydisease, including IL 4R-α, for use in pharmaceutical compositions.Patent application US 20050143333 teaches a series of siRNAs targeted tointerleukins and interleukin receptors, including IL 4R-α. PCTapplication WO 2004045543 teaches algorithms and rational design andselection of functional siRNAs including those targeted to IL 4R-α.Although it is suggested in these publications that the ASOs and siRNAscan be used in pharmaceutical compositions, there are no datademonstrating the efficacy of the compounds in vivo for the prevention,amelioration, and/or treatment of any disease or disorder.

SUMMARY OF THE INVENTION

The invention provides compounds, particularly oligomeric compounds,especially nucleic acid and nucleic acid-like oligomers, which aretargeted to a nucleic acid encoding IL-4R alpha. Preferably, theoligomeric compounds are antisense oligonucleotides targeted to IL 4R-α;particularly human IL 4R-α (GenBank Accession No. X52425.1, entered 26May 1992 (SEQ ID NO. 1); GenBank Accession No. BM738518.1, entered 1Mar. 2002; nucleotides 18636000 to 18689000 of GenBank Accession No.NT_(—)010393.14 entered 19 Feb. 2004, each of which is incorporated byreference), that modulate the expression of IL 4R-α. The compoundscomprise at least a 12 nucleobase portion, preferably at least a 17nucleobase portion of the sequences listed in Table 3, 4 or 5, or are atleast 90% identical to validated target segments, or the sequenceslisted in Table 3, 4, or 5.

The invention provides a method for modulating the expression of IL 4R-αin cells or tissues comprising contacting the cells with at least onecompound of the instant invention, and analyzing the cells forindicators of a decrease in expression of IL 4R-α mRNA and/or protein bydirect measurement of mRNA and/or protein levels, and/or indicators ofpulmonary inflammation and/or airway hyperresponsiveness.

The invention further provides a method for the prevention,amelioration, and/or treatment of pulmonary inflammation and/or airwayhyperresponsiveness comprising administering at least one compound ofthe instant invention to an individual in need of such intervention. Thecompound is preferably administered by aerosol (i.e., topically) to atleast a portion of the respiratory tract. The portion of the respiratorytract selected is dependent upon the location of the inflammation. Forexample, in the case of asthma, the compound is preferably deliveredpredominantly to the lung. In the case of allergic rhinitis, thecompound is preferably delivered predominantly to the nasal cavityand/or sinus. The compound is delivered using any of a number ofstandard delivery devices and methods well known to those skilled in theart, including, but not limited to nebulizers, nasal and pulmonaryinhalers, dry powder inhalers, and metered dose inhalers.

The invention also provides a method of use of the compositions of theinstant invention for the preparation of a medicament for theprevention, amelioration, and/or treatment disease, especially a diseaseassociated with and including at least one indicator of pulmonaryinflammation and/or airway hyperresponsiveness. The medicament ispreferably formulated for aerosol administration to at least a portionof the respiratory tract.

DETAILED DESCRIPTION OF THE INVENTION

Asthma, allergy, and a number of other diseases or conditions related topulmonary inflammation and/or AHR share common inflammatory mediators,including IL 4R-α, the common subunit of IL 4R and IL 13R. Therapeuticinterventions for these diseases or conditions are not completelysatisfactory due to lack of efficacy and/or unwanted side effects of thecompounds. The instant invention provides oligomeric compounds,preferably ASOs, for the prevention, amelioration, and/or treatment ofpulmonary inflammation and/or airway hyperresponsiveness. As usedherein, the term “prevention” means to delay of forestall onset ordevelopment of a condition or disease for a period of time from hours todays, preferably weeks to months. As used herein, the term“amelioration” means a lessening of at least one indicator of theseverity of a condition or disease. The severity of indicators may bedetermined by subjective or objective measures. As used herein,“treatment” means to administer a composition of the invention to effectan alteration or improvement of the disease or condition. Prevention,amelioration, and/or treatment may require administration of multipledoses at regular intervals, or prior to exposure to an agent (e.g., anallergen) to alter the course of the condition or disease. Moreover, asingle agent may be used in a single individual for each prevention,amelioration, and treatment of a condition or disease sequentially, orconcurrently. In a preferred method of the instant invention, the ASOsare delivered by aerosol for topical delivery to the respiratory tract,thereby limiting systemic exposure and reducing potential side effects.

Overview

Disclosed herein are oligomeric compounds, including antisenseoligonucleotides and other antisense compounds for use in modulating theexpression of nucleic acid molecules encoding IL 4R-α This isaccomplished by providing oligomeric compounds that hybridize with oneor more target nucleic acid molecules encoding IL 4R-α. As used herein,the terms “target nucleic acid” and “nucleic acid molecule encoding IL4R-α” have been used for convenience to encompass DNA encoding IL 4R-α,RNA (including pre-mRNA and mRNA or portions thereof) transcribed fromsuch DNA, and also cDNA derived from such RNA. In a preferredembodiment, the target nucleic acid is an mRNA encoding IL 4R-α.

The principle behind antisense technology is that an antisense compoundthat hybridizes to a target nucleic acid, modulates gene expressionactivities such as transcription or translation. This sequencespecificity makes antisense compounds extremely attractive as tools fortarget validation and gene functionalization, as well as therapeutics toselectively modulate the expression of genes involved in disease.Although not limited by mechanism of action, the compounds of theinstant invention are proposed to work by an antisense,non-autocatalytic mechanism.

Compounds

The term “oligomeric compound” refers to a polymeric structure capableof hybridizing to a region of a nucleic acid molecule. This termincludes oligonucleotides, oligonucleosides, oligonucleotide analogs,oligonucleotide mimetics, and chimeric combinations of these. Generally,oligomeric compounds comprise a plurality of monomeric subunits linkedtogether by internucleoside linking groups and/or internucleosidelinkage mimetics. Each of the monomeric subunits comprises a sugar,abasic sugar, modified sugar, or a sugar mimetic, and except for theabasic sugar includes a nucleobase, modified nucleobase or a nucleobasemimetic. Preferred monomeric subunits comprise nucleosides and modifiednucleosides. Oligomeric compounds are routinely prepared linearly butcan be joined or otherwise prepared to be circular. Moreover, branchedstructures are known in the art.

An “antisense compound” or “antisense oligomeric compound” refers to anoligomeric compound that is at least partially complementary to theregion of a target nucleic acid molecule to which it hybridizes andwhich modulates (increases or decreases) its expression. Consequently,while all antisense compounds can be said to be oligomeric compounds,not all oligomeric compounds are antisense compounds. An “antisenseoligonucleotide” is an antisense compound that is a nucleic acid-basedoligomer. An antisense oligonucleotide can, in some cases, include oneor more chemical modifications to the sugar, base, and/orinternucleoside linkages. Nonlimiting examples of oligomeric compoundsinclude primers, probes, antisense compounds, antisenseoligonucleotides, external guide sequence (EGS) oligonucleotides,alternate splicers, and siRNAs. As such, these compounds can beintroduced in the form of single-stranded, double-stranded, circular,branched or hairpins and can contain structural elements such asinternal or terminal bulges or loops. Oligomeric double-strandedcompounds can be two strands hybridized to form double-strandedcompounds or a single strand with sufficient self complementarity toallow for hybridization and formation of a fully or partiallydouble-stranded compound. The compounds of the instant invention are notauto-catalytic. As used herein, “auto-catalytic” means a compound hasthe ability to promote cleavage of the target RNA in the absence ofaccessory factors, e.g. proteins.

In one embodiment of the invention, the oligomeric compound is anantisense compound comprising a single stranded oligonucleotide. In someembodiments of the invention the oligomeric compound contains chemicalmodifications. In a preferred embodiment, the antisense compound is asingle stranded, chimeric oligonucleotide wherein the modifications ofsugars, bases, and internucleoside linkages are independently selected.

The oligomeric compounds in accordance with this invention may comprisean oligomeric compound from about 12 to about 35 nucleobases (i.e. fromabout 12 to about 35 linked nucleosides). In other words, asingle-stranded compound of the invention comprises from about 12 toabout 35 nucleobases, and a double-stranded antisense compound of theinvention (such as a siRNA, for example) comprises two strands, each ofwhich is from about 12 to about 35 nucleobases. Contained within theoligomeric compounds of the invention (whether single or double strandedand on at least one strand) are antisense portions. The “antisenseportion” is that part of the oligomeric compound that is designed towork by one of the aforementioned antisense mechanisms. One of ordinaryskill in the art will appreciate that this comprehends antisenseportions of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, or 35 nucleobases.

In one embodiment, the antisense compounds of the invention haveantisense portions of 12 to 35 nucleobases. It is understood that theantisense portion may be about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 nucleobases inlength.

Antisense compounds 12 to 35 nucleobases in length comprising a stretchof at least eight (8), preferably at least 12, more preferably at least17 consecutive nucleobases selected from within the illustrativeantisense compounds are considered to be suitable antisense compounds aswell.

Compounds of the invention include oligonucleotide sequences thatcomprise at least the 8 consecutive nucleobases from the 5′-terminus ofone of the illustrative antisense compounds (the remaining nucleobasesbeing a consecutive stretch of the same oligonucleotide beginningimmediately upstream of the 5′-terminus of the antisense compound whichis specifically hybridizable to the target nucleic acid and continuinguntil the oligonucleotide contains about 12 to 35 nucleobases). Othercompounds are represented by oligonucleotide sequences that comprise atleast the 8 consecutive nucleobases from the 3′-terminus of one of theillustrative antisense compounds (the remaining nucleobases being aconsecutive stretch of the same oligonucleotide beginning immediatelydownstream of the 3′-terminus of the antisense compound which isspecifically hybridizable to the target nucleic acid and continuinguntil the oligonucleotide contains about 12 to about 35 nucleobases). Itis also understood that compounds may be represented by oligonucleotidesequences that comprise at least 8 consecutive nucleobases from aninternal portion of the sequence of an illustrative compound, and mayextend in either or both directions until the oligonucleotide containsabout 12 to about 35 nucleobases.

Modifications can be made to the compounds of the instant invention andmay include conjugate groups attached to one of the termini, selectednucleobase positions, sugar positions or to one of the internucleosidelinkages. Possible modifications include, but are not limited to, 2′-Fand 2′-OMethyl sugar modifications, inverted abasic caps,deoxynucleobases, and nucleobase analogs such as locked nucleic acids(LNA).

In one embodiment of the invention, double-stranded antisense compoundsencompass short interfering RNAs (siRNAs). As used herein, the term“siRNA” is defined as a double-stranded compound having a first andsecond strand, each strand having a central portion and two independentterminal portions. The central portion of the first strand iscomplementary to the central portion of the second strand, allowinghybridization of the strands. The terminal portions are independently,optionally complementary to the corresponding terminal portion of thecomplementary strand. The ends of the strands may be modified by theaddition of one or more natural or modified nucleobases to form anoverhang. In one nonlimiting example, the first strand of the siRNA isantisense to the target nucleic acid, while the second strand iscomplementary to the first strand. Once the antisense strand is designedto target a particular nucleic acid target, the sense strand of thesiRNA can then be designed and synthesized as the complement of theantisense strand and either strand may contain modifications oradditions to either terminus. For example, in one embodiment, bothstrands of the siRNA duplex would be complementary over the centralnucleobases, each having overhangs at one or both termini. It ispossible for one end of a duplex to be blunt and the other to haveoverhanging nucleobases. In one embodiment, the number of overhangingnucleobases is from 1 to 6 on the 3′ end of each strand of the duplex.In another embodiment, the number of overhanging nucleobases is from 1to 6 on the 3′ end of only one strand of the duplex. In a furtherembodiment, the number of overhanging nucleobases is from 1 to 6 on oneor both 5′ ends of the duplexed strands. In another embodiment, thenumber of overhanging nucleobases is zero. In a preferred embodiment,each of the strands is 19 nucleobases in length, fully hybridizable withthe complementary strand, and includes no overhangs.

Each strand of the siRNA duplex may be from about 12 to about 35nucleobases. In a preferred embodiment, each strand of the siRNA duplexis about 17 to about 25 nucleobases. The central complementary portionmay be from about 12 to about 35 nucleobases in length. In a preferredembodiment, the central complimentary portion is about 17 to about 25nucleobases in length. It is understood that each the strand of thesiRNA duplex and the central complementary portion may be about 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, or 35 nucleobases in length. The terminal portions can befrom 1 to 6 nucleobases. It is understood that the terminal portions canbe about 1, 2, 3, 4, 5, or 6 nucleobases in length. The siRNAs may alsohave no terminal portions. The two strands of an siRNA can be linkedinternally leaving free 3′ or 5′ termini, or can be linked to form acontinuous hairpin structure or loop. The hairpin structure may containan overhang on either the 5′ or 3′ terminus producing an extension ofsingle-stranded character.

Double-stranded compounds can be made to include chemical modificationsas discussed herein.

Chemical Modifications

As is known in the art, a nucleoside is a base-sugar combination. Thebase portion of the nucleoside is normally a heterocyclic base(sometimes referred to as a “nucleobase” or simply a “base”). The twomost common classes of such heterocyclic bases are the purines and thepyrimidines. Nucleotides are nucleosides that further include aphosphate group covalently linked to the sugar portion of thenucleoside. For those nucleosides that include a pentofuranosyl sugar,the phosphate group can be linked to the 2′, 3′ or 5′ hydroxyl moiety ofthe sugar. In forming oligonucleotides, the phosphate groups covalentlylink adjacent nucleosides to one another to form a linear polymericcompound. In turn, the respective ends of this linear polymeric compoundcan be further joined to form a circular compound. Withinoligonucleotides, the phosphate groups are commonly referred to asforming the internucleoside backbone of the oligonucleotide. The normallinkage or backbone of RNA and DNA is a 3′ to 5′ phosphodiester linkage.It is often preferable to include chemical modifications inoligonucleotides to alter their activity. Chemical modifications canalter oligonucleotide activity by, for example: increasing affinity ofan antisense oligonucleotide for its target RNA, increasing nucleaseresistance, and/or altering the pharmacokinetics of the oligonucleotide.The use of chemistries that increase the affinity of an oligonucleotidefor its target can allow for the use of shorter oligonucleotidecompounds.

The term “nucleobase” or “heterocyclic base moiety” as used herein,refers to the heterocyclic base portion of a nucleoside. In general, anucleobase is any group that contains one or more atom or groups ofatoms capable of hydrogen bonding to a base of another nucleic acid. Inaddition to “unmodified” or “natural” nucleobases such as the purinenucleobases adenine (A) and guanine (G), and the pyrimidine nucleobasesthymine (T), cytosine (C) and uracil (U), many modified nucleobases ornucleobase mimetics known to the art skilled are amenable to the presentinvention. The terms modified nucleobase and nucleobase mimetic canoverlap but generally a modified nucleobase refers to a nucleobase thatis fairly similar in structure to the parent nucleobase such as forexample a 7-deaza purine or a 5-methyl cytosine whereas a nucleobasemimetic would include more complicated structures such as for example atricyclic phenoxazine nucleobase mimetic. Methods for preparation of theabove noted modified nucleobases are well known to those skilled in theart.

Oligomeric compounds of the present invention may also contain one ormore nucleosides having modified sugar moieties. The furanosyl sugarring of a nucleoside can be modified in a number of ways including, butnot limited to, addition of a substituent group, bridging of twonon-geminal ring atoms to form a bicyclic nucleic acid (BNA) andsubstitution of an atom or group such as —S—, —N(R)— or —C(R₁)(R₂) forthe ring oxygen at the 4′-position, Modified sugar moieties are wellknown and can be used to alter, typically increase, the affinity of theoligomeric compound for its target and/or increase nuclease resistance.A representative list of preferred modified sugars includes but is notlimited to bicyclic modified sugars (BNA's), including LNA and ENA(4′-(CH₂)₂—O-2′ bridge); and substituted sugars, especially2′-substituted sugars having a 2′-F, 2′-OCH₂ or a 2′-O(CH₂)₂—OCH₃substituent group. Sugars can also be replaced with sugar mimetic groupsamong others. Methods for the preparations of modified sugars are wellknown to those skilled in the art.

The present invention includes internucleoside linking groups that linkthe nucleosides or otherwise modified monomer units together therebyforming an oligomeric compound. The two main classes of internucleosidelinking groups are defined by the presence or absence of a phosphorusatom. Representative phosphorus containing internucleoside linkagesinclude, but are not limited to, phosphodiesters, phosphotriesters,methylphosphonates, phosphoramidate, and phosphorothioates.Representative non-phosphorus containing internucleoside linking groupsinclude, but are not limited to, methylenemethylimino(—CH₂—N(CH₃)—O—CH₂—), thiodiester (—O—C(O)—S—), thionocarbamate(—O—C(O)(NH)—S—); siloxane (—O—Si(H)₂—O—); and N,N′-dimethylhydrazine(—CH₂—N(CH₃)—N(CH₃)—). Oligomeric compounds having non-phosphorusinternucleoside linking groups are referred to as oligonucleosides.Modified internucleoside linkages, compared to natural phosphodiesterlinkages, can be used to alter, typically increase, nuclease resistanceof the oligomeric compound. Internucleoside linkages having a chiralatom can be prepared racemic, chiral, or as a mixture. Representativechiral internucleoside linkages include, but are not limited to,alkylphosphonates and phosphorothioates. Methods of preparation ofphosphorous-containing and non-phosphorous-containing linkages are wellknown to those skilled in the art.

As used herein the term “mimetic” refers to groups that are substitutedfor a sugar, a nucleobase, and/or internucleoside linkage. Mimetics aregroups that are structurally quite different (not simply a modification)but functionally similar to the linked nucleosides of oligonucleotides.Generally, a mimetic is used in place of the sugar orsugar-internucleoside linkage combination, and the nucleobase ismaintained for hybridization to a selected target. Representativeexamples of a sugar mimetic include, but are not limited to,cyclohexenyl or morpholino. Representative examples of a mimetic for asugar-internucleoside linkage combination include, but are not limitedto, peptide nucleic acids (PNA) and morpholino groups linked byuncharged achiral linkages. In some instances a mimetic is used in placeof the nucleobase. Representative nucleobase mimetics are well known inthe art and include, but are not limited to, tricyclic phenoxazineanalogs and universal bases (Berger et al., Nuc Acid Res. 2000,28:2911-14, incorporated herein by reference). Methods of synthesis ofsugar, nucleoside and nucleobase mimetics are well known to thoseskilled in the art.

As used herein the term “nucleoside” includes, nucleosides, abasicnucleosides, modified nucleosides, and nucleosides having mimetic basesand/or sugar groups.

In the context of this invention, the term “oligonucleotide” refers toan oligomeric compound which is an oligomer or polymer of ribonucleicacid (RNA) or deoxyribonucleic acid (DNA). This term includesoligonucleotides composed of naturally- and non-naturally-occurringnucleobases, sugars and covalent internucleoside linkages, possiblyfurther including non-nucleic acid conjugates.

The present invention provides compounds having reactive phosphorusgroups useful for forming internucleoside linkages including for examplephosphodiester and phosphorothioate internucleoside linkages. Methods ofpreparation and/or purification of precursors or olgomeric compounds ofthe instant invention are not a limitation of the compositions ormethods of the invention. Methods for synthesis and purification of DNA,RNA, and the oligomeric compounds of the instant invention are wellknown to those skilled in the art.

As used herein the term “chimeric oligomeric compound” refers to anoligomeric compound having at least one sugar, nucleobase and/orinternucleoside linkage that is differentially modified as compared tothe other sugars, nucleobases and internucleoside linkages within thesame oligomeric compound. The remainder of the sugars, nucleobases andinternucleoside linkages can be independently modified or unmodifiedprovided that they are distinguishable from the differentially modifiedmoiety or moieties. In general a chimeric oligomeric compound will havemodified nucleosides that can be in isolated positions or groupedtogether in regions that will define a particular motif. Any combinationof modifications and or mimetic groups can comprise a chimericoligomeric compound of the present invention.

Chimeric oligomeric compounds typically contain at least one regionmodified so as to confer increased resistance to nuclease degradation,increased cellular uptake, and/or increased binding affinity for thetarget nucleic acid. An additional region of the oligomeric compound mayserve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNAhybrids. By way of example, RNase H is a cellular endonuclease thatcleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H,therefore, results in cleavage of the RNA target, thereby greatlyenhancing the efficiency of inhibition of gene expression. Consequently,comparable results can often be obtained with shorter oligomericcompounds when chimeras are used, compared to for examplephosphorothioate deoxyoligonucleotides hybridizing to the same targetregion. Cleavage of the RNA target can be routinely detected by gelelectrophoresis and, if necessary, associated nucleic acid hybridizationtechniques known in the art.

Certain chimeric as well as non-chimeric oligomeric compounds can befurther described as having a particular motif. As used in the presentinvention the term “motif” refers to the orientation of modified sugarmoieties and/or sugar mimetic groups in an oligomeric compound relativeto like or differentially modified or unmodified nucleosides. As used inthe present invention, the terms “sugars”, “sugar moieties” and “sugarmimetic groups’ are used interchangeably. Such motifs include, but arenot limited to, gapped motifs, alternating motifs, fully modifiedmotifs, hemimer motifs, blockmer motifs, and positionally modifiedmotifs. The sequence and the structure of the nucleobases and type ofinternucleoside linkage is not a factor in determining the motif of anoligomeric compound.

As used in the present invention the term “gapped motif” refers to anoligomeric compound comprising a contiguous sequence of nucleosides thatis divided into 3 regions, an internal region (gap) flanked by twoexternal regions (wings). The regions are differentiated from each otherat least by having differentially modified sugar groups that comprisethe nucleosides. In some embodiments, each modified region is uniformlymodified (e.g. the modified sugar groups in a given region areidentical); however, other motifs can be applied to regions. Forexample, the wings in a gapmer could have an alternating motif. Theinternal region or the gap may, in some instances, comprise uniformunmodified β-D-ribonucleosides or β-D-deoxyribonucleosides or can be asequence of nucleosides having uniformly modified sugars. Thenucleosides located in the gap of a gapped oligomeric compound havesugar moieties that are different than the modified sugar moieties ineach of the wings.

As used in the present invention the term “alternating motif” refers toan oligomeric compound comprising a contiguous sequence of nucleosidescomprising two differentially sugar modified nucleosides that alternatefor essentially the entire sequence of the oligomeric compound, or foressentially the entire sequence of a region of an oligomeric compound.The pattern of alternation can be described by the formula:5′-A(-L-B-L-A)n(-L-B)nn-3′ where A and B are nucleosides differentiatedby having at least different sugar groups, each L is an internucleosidelinking group, nn is preferably 0 or 1 and n is preferably from about 5to about 11; however, the number may be larger than about 11. Thisformula also allows for even and odd lengths for alternating oligomericcompounds wherein the 3′ and 5′-terminal nucleosides are the same (odd)or different (even).

As used in the present invention the term “fully modified motif” refersto an oligomeric compound comprising a contiguous sequence ofnucleosides wherein essentially each nucleoside is a sugar modifiednucleoside having uniform modification.

As used in the present invention the term “hemimer motif” refers to asequence of nucleosides that have uniform sugar moieties (identicalsugars, modified or unmodified) and wherein one of the 5′-end or the3′-end has a sequence of from 2 to 12 nucleosides that are sugarmodified nucleosides that are different from the other nucleosides inthe hemimer modified oligomeric compound. An example of a typicalhemimer is an oligomeric compound comprising β-D-deoxyribonucleosideshaving a contiguous sequence of sugar modified nucleosides at one of thetermini.

As used in the present invention the term “blockmer motif” refers to asequence of nucleosides that have uniform sugars (identical sugars,modified or unmodified) that is internally interrupted by a block ofsugar modified nucleosides that are uniformly modified and wherein themodification is different from the other nucleosides. In one aspect ofthe present invention oligomeric compounds having a blockmer motifcomprise a sequence of β-D-deoxyribonucleosides having one internalblock of from 2 to 6 sugar modified nucleosides. The internal blockregion can be at any position within the oligomeric compound as long asit is not at one of the termini which would then make it a hemimer.Methods of preparation of chimeric oligonucleotide compounds are wellknown to those skilled in the art.

As used in the present invention the term “positionally modified motif”comprises all other motifs. Methods of preparation of positionallymodified oligonucleotide compounds are well known to those skilled inthe art.

The compounds described herein contain one or more asymmetric centersand thus give rise to enantiomers, diastereomers, and otherstereoisomeric configurations that may be defined, in terms of absolutestereochemistry, as (R) or (S), u or 1, or as (D) or (L) such as foramino acids et al. The present invention is meant to include all suchpossible isomers, as well as their racemic and optically pure forms.

In one aspect of the present invention oligomeric compounds are modifiedby covalent attachment of one or more conjugate groups. Conjugate groupsmay be attached by reversible or irreversible attachments. Conjugategroups may be attached directly to oligomeric compounds or by use of alinker. Linkers may be mono- or bifunctional linkers. Such attachmentmethods and linkers are well known to those skilled in the art. Ingeneral, conjugate groups are attached to oligomeric compounds to modifyone or more properties. Such considerations are well known to thoseskilled in the art.

Oligomer Synthesis

Oligomerization of modified and unmodified nucleosides can be routinelyperformed according to literature procedures for DNA (Protocols forOligonucleotides and Analogs, Ed. Agrawal (1993), Humana Press) and/orRNA (Scaringe, Methods (2001), 23, 206-217. Gait et al., Applications ofChemically synthesized RNA in RNA: Protein Interactions, Ed. Smith(1998), 1-36. Gallo et al., Tetrahedron (2001), 57, 5707-5713).

Oligomeric compounds of the present invention can be conveniently androutinely made through the well-known technique of solid phasesynthesis. Equipment for such synthesis is sold by several vendorsincluding, for example, Applied Biosystems (Foster City, Calif.). Anyother means for such synthesis known in the art may additionally oralternatively be employed. It is well known to use similar techniques toprepare oligonucleotides such as the phosphorothioates and alkylatedderivatives. The invention is not limited by the method of oligomersynthesis.

Oligomer Purification and Analysis

Methods of oligonucleotide purification and analysis are known to thoseskilled in the art. Analysis methods include capillary electrophoresis(CE) and electrospray-mass spectroscopy. Such synthesis and analysismethods can be performed in multi-well plates. The method of theinvention is not limited by the method of oligomer purification.

Hybridization

“Hybridization” means the pairing of complementary strands of oligomericcompounds. While not limited to a particular mechanism, the most commonmechanism of pairing involves hydrogen bonding, which may beWatson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, betweencomplementary nucleoside or nucleotide bases (nucleobases) of thestrands of oligomeric compounds. For example, adenine and thymine arecomplementary nucleobases which pair through the formation of hydrogenbonds. Hybridization can occur under varying circumstances.

An oligomeric compound is specifically hybridizable when there is asufficient degree of complementarity to avoid non-specific binding ofthe oligomeric compound to non-target nucleic acid sequences underconditions in which specific binding is desired, i.e., underphysiological conditions in the case of in vivo assays or therapeutictreatment, and under conditions in which assays are performed in thecase of in vitro assays.

“Stringent hybridization conditions” or “stringent conditions” refers toconditions under which an oligomeric compound will hybridize to itstarget sequence, but to a minimal number of other sequences. Stringentconditions are sequence-dependent and will be different in differentcircumstances, and “stringent conditions” under which oligomericcompounds hybridize to a target sequence are determined by the natureand composition of the oligomeric compounds and the assays in which theyare being investigated.

“Complementarity,” as used herein, refers to the capacity for precisepairing between two nucleobases on one or two oligomeric compoundstrands. For example, if a nucleobase at a certain position of anantisense compound is capable of hydrogen bonding with a nucleobase at acertain position of a target nucleic acid, then the position of hydrogenbonding between the oligonucleotide and the target nucleic acid isconsidered to be a complementary position. The oligomeric compound andthe further DNA or RNA are complementary to each other when a sufficientnumber of complementary positions in each molecule are occupied bynucleobases which can hydrogen bond with each other. Thus, “specificallyhybridizable” and “complementary” are terms which are used to indicate asufficient degree of precise pairing or complementarity over asufficient number of nucleobases such that stable and specific bindingoccurs between the oligomeric compound and a target nucleic acid.

Identity

Oligomeric compounds, or a portion thereof, may have a defined percentidentity to a SEQ ID NO, or a compound having a specific Isis number. Asused herein, a sequence is identical to the sequence disclosed herein ifit has the same nucleobase pairing ability. For example, a RNA whichcontains uracil in place of thymidine in the disclosed sequences of theinstant invention would be considered identical as they both pair withadenine. Similarly, a G-clamp modified heterocyclic base would beconsidered identical to a cytosine or a 5-Me cytosine in the sequencesof the instant application as it pairs with a guanine. This identity maybe over the entire length of the oligomeric compound, or in a portion ofthe oligomeric compound (e.g., nucleobases 1-20 of a 27-mer may becompared to a 20-mer to determine percent identity of the oligomericcompound to the SEQ ID NO.) It is understood by those skilled in the artthat an oligonucleotide need not have an identical sequence to thosedescribed herein to function similarly to the oligonucleotides describedherein. Shortened (i.e., deleted, and therefore non-identical) versionsof oligonucleotides taught herein, or non-identical (e.g., one basereplaced with another with non-identical nucleobase pairing, or abasicsite) versions of the oligonucleotides taught herein fall within thescope of the invention. Percent identity is calculated according to thenumber of bases that have identical base pairing corresponding to theSEQ ID NO or compound to which it is being compared. The non-identicalbases may be adjacent to each other, dispersed through out theoligonucleotide, or both.

For example, a 16-mer having the same sequence as nucleobases 2-17 of a20-mer is 80% identical to the 20-mer. Alternatively, a 20-mercontaining four nucleobases not identical to the 20-mer is also 80%identical to the 20-mer. A 14-mer having the same sequence asnucleobases 1-14 of an 18-mer is 78% identical to the 18-mer. Suchcalculations are well within the ability of those skilled in the art.

The percent identity is based on the percent of nucleobases in theoriginal sequence present in a portion of the modified sequence.Therefore, a 30 nucleobase oligonucleotide comprising the full sequenceof a 20 nucleobase SEQ ID NO would have a portion of 100% identity withthe 20 nucleobase SEQ ID NO while further comprising an additional 10nucleobase portion. In the context of the invention, the full length ofthe modified sequence may constitute a single portion. In a preferredembodiment, the oligonucleotides of the instant invention are at leastabout 80%, more preferably at least about 85%, most preferably at leastabout 90% identical to the active target segments and/oroligonucleotides presented herein.

It is well known by those skilled in the art that it is possible toincrease or decrease the length of an antisense oligonucleotide and/orintroduce mismatch bases without eliminating activity. For example, inWoolf et al. (Proc. Natl. Acad. Sci. USA 89:7305-7309. 1992,incorporated herein by reference), a series of oligomers 13-25nucleobases in length were tested for their ability to induce cleavageof a target RNA in an oocyte injection model. Oligonucleotides 25nucleobases in length with 8 or 11 mismatch bases near the ends of theoligonucleotide were able to direct specific cleavage of the targetmRNA, albeit to a lesser extent than the oligonucleotide that containedno mismatches. Similarly, target specific cleavage was achieved using a13 nucleobase oligomer, including those with 1 or 3 mismatches. Maherand Dolnick (Nuc. Acid. Res. 16:3341-3358.1988, incorporated herein byreference) tested a series of tandem 14 nucleobase oligonucleotides, anda 28 and 42 nucleobase oligonucleotide comprised of the sequence of twoor three of the tandem oligonucleotides, respectively, for their abilityto arrest translation of human DHFR in a rabbit reticulocyte assay. Eachof the three 14 nucleobase oligonucleotides alone were able to inhibittranslation, albeit at a more modest level, than the 28 or 42 nucleobaseoligonucleotide.

Target Nucleic Acids

“Targeting” an oligomeric compound to a particular target nucleic acidmolecule can be a multistep process. The process usually begins with theidentification of a target nucleic acid whose expression is to bemodulated. For example, the target nucleic acid can be a cellular gene(or mRNA transcribed from the gene) whose expression is associated witha particular disorder or disease state, or a nucleic acid molecule froman infectious agent. As disclosed herein, the target nucleic acidencodes IL-4R alpha.

Target Regions, Segments, and Sites

The targeting process usually also includes determination of at leastone target region, segment, or site within the target nucleic acid forthe antisense interaction to occur such that the desired effect, e.g.,modulation of expression, will result. “Region” is defined as a portionof the target nucleic acid having at least one identifiable structure,function, or characteristic. Target regions include, but are not limitedto translation initiation and termination regions, coding regions, openreading frames, introns, exons, 3′-untranslated regions (3′-UTR), and5′-untranslated regions (5′-UTR). Within regions of target nucleic acidsare segments. “Segments” are defined as smaller or sub-portions ofregions within a target nucleic acid such as stop codons and startcodons. “Sites,” as used in the present invention, are defined as uniquenucleobase positions within a target nucleic acid such as splicejunctions. Such regions, segments, and sites are well known to thoseskilled in the art.

Variants

It is also known in the art that alternative RNA transcripts can beproduced from the same genomic region of DNA. These alternativetranscripts are generally known as “variants.” More specifically,“pre-mRNA variants” are transcripts produced from the same genomic DNAthat differ from other transcripts produced from the same genomic DNA ineither their start or stop position and contain both intronic and exonicsequence. Variants can result in mRNA variants including, but notlimited to, those with alternate splice junctions, or alternateinitiation and termination codons. Variants in genomic and mRNAsequences can result in disease. Oligonucleotides to such variants arewithin the scope of the instant invention.

Target Names, Synonyms, Features

In accordance with the present invention are compositions and methodsfor modulating the expression of IL 4R-α (also known as Interleukin 4alpha receptor; CD 124; IL-4Ra; interleukin 4 receptor alpha chain).Table 1 lists the GenBank accession numbers of sequences correspondingto nucleic acid molecules encoding IL 4R-α (nt=nucleotide), the date theversion of the sequence was entered in GenBank, and the correspondingSEQ ID NO in the instant application, when assigned, each of which isincorporated herein by reference.

TABLE 1 Gene Targets SEQ ID Species Genbank # Genbank Date NO HumanBM738518.1 1 Mar. 2002 Human nt 18636000 to 18689000 of 19 Feb. 2004NT_010393.14 Human X52425.1 26 May 1992 1 Mouse AF000304.1 1 Dec. 1997Mouse assembled from M64868.1 Both 6 May 1996 and M64879.1 MouseBB867141.1 9 Jul. 2003 Mouse BC012309.1 3 Jan. 2005 Mouse M27959.1 16Sep. 1994 Mouse M27960.1 12 Jun. 1993 2 Mouse M29854.1 12 Jun. 1993

Modulation of Target Expression

Modulation of expression of a target nucleic acid can be achievedthrough alteration of any number of nucleic acid (DNA or RNA) functions.“Modulation” means a perturbation of function, for example, either anincrease (stimulation or induction) or a decrease (inhibition orreduction) in expression. As another example, modulation of expressioncan include perturbing splice site selection of pre-mRNA processing“Expression” includes all the functions by which a gene's codedinformation is converted into structures present and operating in acell. These structures include the products of transcription andtranslation. “Modulation of expression” means the perturbation of suchfunctions. The functions of RNA to be modulated can includetranslocation functions, which include, but are not limited to,translocation of the RNA to a site of protein translation, translocationof the RNA to sites within the cell which are distant from the site ofRNA synthesis, and translation of protein from the RNA. RNA processingfunctions that can be modulated include, but are not limited to,splicing of the RNA to yield one or more RNA species, capping of theRNA, 3′ maturation of the RNA and catalytic activity or complexformation involving the RNA which may be engaged in or facilitated bythe RNA. Modulation of expression can result in the increased level ofone or more nucleic acid species or the decreased level of one or morenucleic acid species, either temporally or by net steady state level.One result of such interference with target nucleic acid function ismodulation of the expression of IL 4R-α. Thus, in one embodimentmodulation of expression can mean increase or decrease in target RNA orprotein levels. In another embodiment modulation of expression can meanan increase or decrease of one or more RNA splice products, or a changein the ratio of two or more splice products.

The effect of oligomeric compounds of the present invention on targetnucleic acid expression can be tested in any of a variety of cell typesprovided that the target nucleic acid is present at measurable levels.The effect of oligomeric compounds of the present invention on targetnucleic acid expression can be routinely determined using, for example,PCR or Northern blot analysis. Cell lines are derived from both normaltissues and cell types and from cells associated with various disorders(e.g. hyperproliferative disorders). Cell lines derived from multipletissues and species can be obtained from American Type CultureCollection (ATCC, Manassas, Va.) and are well known to those skilled inthe art. Primary cells, or those cells which are isolated from an animaland not subjected to continuous culture, can be prepared according tomethods known in the art or obtained from various commercial suppliers.Additionally, primary cells include those obtained from donor humansubjects in a clinical setting (i.e. blood donors, surgical patients).Primary cells prepared by methods known in the art.

Assaying Modulation of Expression

Modulation of IL 4R-α expression can be assayed in a variety of waysknown in the art. IL 4R-α mRNA levels can be quantitated by, e.g.,Northern blot analysis, competitive polymerase chain reaction (PCR), orreal-time PCR. RNA analysis can be performed on total cellular RNA orpoly(A)+ mRNA by methods known in the art. Methods of RNA isolation aretaught in, for example, Ausubel, F. M. et al., Current Protocols inMolecular Biology, Volume 1, pp. 4.1.1-4.2.9 and 4.5.1-4.5.3, John Wiley& Sons, Inc., 1993.

Northern blot analysis is routine in the art and is taught in, forexample, Ausubel, F. M. et al., Current Protocols in Molecular Biology,Volume 1, pp. 4.2.1-4.2.9, John Wiley & Sons, Inc., 1996. Real-timequantitative (PCR) can be conveniently accomplished using thecommercially available ABI PRISM™ 7700 Sequence Detection System,available from PE-Applied Biosystems, Foster City, Calif. and usedaccording to manufacturers instructions. The method of analysis ofmodulation of RNA levels is not a imitation of the instant invention.

Levels of a protein encoded by IL 4R-α can be quantitated in a varietyof ways well known in the art, such as immunoprecipitation, Western blotanalysis (immunoblotting), ELISA or fluorescence-activated cell sorting(FACS). Antibodies directed to a protein encoded by IL 4R-α can beidentified and obtained from a variety of sources, such as the MSRScatalog of antibodies (Aerie Corporation, Birmingham, Mich.), or can beprepared via conventional antibody generation methods. Methods forpreparation of polyclonal antisera are taught in, for example, Ausubel,F. M. et al., Current Protocols in Molecular Biology, Volume 2, pp.11.12.1-11.12.9, John Wiley & Sons, Inc., 1997. Preparation ofmonoclonal antibodies is taught in, for example, Ausubel, F. M. et al.,Current Protocols in Molecular Biology, Volume 2, pp. 11.4.1-11.11.5,John Wiley & Sons, Inc., 1997.

Immunoprecipitation methods are standard in the art and can be found at,for example, Ausubel, F. M. et al., Current Protocols in MolecularBiology, Volume 2, pp. 10.16.1-10.16.11, John Wiley & Sons, Inc., 1998.Western blot (immunoblot) analysis is standard in the art and can befound at, for example, Ausubel, F. M. et al., Current Protocols inMolecular Biology, Volume 2, pp. 10.8.1-10.8.21, John Wiley & Sons,Inc., 1997. Enzyme-linked immunosorbent assays (ELISA) are standard inthe art and can be found at, for example, Ausubel, F. M. et al., CurrentProtocols in Molecular Biology, Volume 2, pp. 11.2.1-11.2.22, John &Sons, Inc., 1991.

Validated Target Segments

The locations on the target nucleic acid to which active oligomericcompounds hybridize are herein below referred to as “validated targetsegments.” As used herein the term “validated target segment” is definedas at least an 8-nucleobase portion of a target region, preferably atleast a 12-nucleobase portion of a target region, to which an activeoligomeric compound is targeted. While not wishing to be bound bytheory, it is presently believed that these target segments representportions of the target nucleic acid which are accessible forhybridization.

Target segments can include DNA or RNA sequences that comprise at leastthe 8, preferably 12 consecutive nucleobases from the 5′-terminus of avalidated target segment (the remaining nucleobases being a consecutivestretch of the same DNA or RNA beginning immediately upstream of the5′-terminus of the target segment and continuing until the DNA or RNAcontains about 12 to about 35 nucleobases). Similarly validated targetsegments are represented by DNA or RNA sequences that comprise at leastthe 8, preferably 12 consecutive nucleobases from the 3′-terminus of avalidated target segment (the remaining nucleobases being a consecutivestretch of the same DNA or RNA beginning immediately downstream of the3′-terminus of the target segment and continuing until the DNA or RNAcontains about 12 to about 35 nucleobases). It is also understood that avalidated oligomeric target segment can be represented by DNA or RNAsequences that comprise at least 8, preferably 12 consecutivenucleobases from an internal portion of the sequence of a validatedtarget segment, and can extend in either or both directions until theoligonucleotide contains about 12 to about 35 nucleobases.

Screening for Modulator Oligomeric Compounds

In another embodiment, the validated target segments identified hereincan be employed in a screen for additional compounds that modulate theexpression of IL 4R-α. “Modulators” are those compounds that modulatethe expression of IL 4R-α and which comprise at least an 8-nucleobaseportion which is complementary to a validated target segment. Thescreening method comprises the steps of contacting a validated targetsegment of a nucleic acid molecule encoding IL 4R-α with one or morecandidate modulators, and selecting for one or more candidate modulatorswhich perturb the expression of a nucleic acid molecule encoding IL4R-α. Once it is shown that the candidate modulator or modulators arecapable of modulating the expression of a nucleic acid molecule encodingIL 4R-α; the modulator can then be employed in further investigativestudies of the function of IL 4R-α, or for use as a research,diagnostic, or therapeutic agent.

Modulator compounds of IL 4R-α can also be identified or furtherinvestigated using one or more phenotypic assays, each having measurableendpoints predictive of efficacy in the treatment of a particulardisease state or condition. Phenotypic assays, kits and reagents fortheir use are well known to those skilled in the art.

Kits, Research Reagents, and Diagnostics

The oligomeric compounds of the present invention can be utilized fordiagnostics, and as research reagents and kits. Furthermore, antisensecompounds, which are able to inhibit gene expression with specificity,are often used by those of ordinary skill to elucidate the function ofparticular genes or to distinguish between functions of various membersof a biological pathway.

For use in kits and diagnostics, the oligomeric compounds of the presentinvention, either alone or in combination with other compounds ortherapeutics, can be used as tools in differential and/or combinatorialanalyses to elucidate expression patterns of a portion or the entirecomplement of genes expressed within cells and tissues. Methods of geneexpression analysis are well known to those skilled in the art.

Therapeutics

Compounds of the invention can be used to modulate the expression of IL4R-α in an animal, such as a human. In one non-limiting embodiment, themethods comprise the step of administering to said animal an effectiveamount of an antisense compound that inhibits expression of IL 4R-α. Inone embodiment, the antisense compounds of the present inventioneffectively inhibit the levels or function of IL 4R-α RNA. Becausereduction in IL 4R-α mRNA levels can lead to alteration in IL 4R-αprotein products of expression as well, such resultant alterations canalso be measured. Antisense compounds of the present invention thateffectively inhibit the level or function of IL 4R-α RNA or proteinproducts of expression is considered an active antisense compound. Inone embodiment, the antisense compounds of the invention inhibit theexpression of IL 4R-α causing a reduction of RNA by at least 10%, by atleast 20%, by at least 25%, by at least 30%, by at least 40%, by atleast 50%, by at least 60%, by at least 70%, by at least 75%, by atleast 80%, by at least 85%, by at least 90%, by at least 95%, by atleast 98%, by at least 99%, or by 100%.

For example, the reduction of the expression of IL 4R-α can be measuredin a bodily fluid, tissue or organ of the animal. Methods of obtainingsamples for analysis, such as body fluids (e.g., sputum), tissues (e.g.,biopsy), or organs, and methods of preparation of the samples to allowfor analysis are well known to those skilled in the art. Methods foranalysis of RNA and protein levels are discussed above and are wellknown to those skilled in the art. The effects of treatment can beassessed by measuring biomarkers associated with the target geneexpression in the aforementioned fluids, tissues or organs, collectedfrom an animal contacted with one or more compounds of the invention, byroutine clinical methods known in the art. These biomarkers include butare not limited to: liver transaminases, bilirubin, albumin, blood ureanitrogen, creatine and other markers of kidney and liver function;interleukins, tumor necrosis factors, intracellular adhesion molecules,C-reactive protein and other markers of inflammation.

The compounds of the present invention can be utilized in pharmaceuticalcompositions by adding an effective amount of a compound to a suitablepharmaceutically acceptable diluent or carrier. Acceptable carriers anddilutents are well known to those skilled in the art. Selection of adilutent or carrier is based on a number of factors, including, but notlimited to, the solubility of the compound and the route ofadministration. Such considerations are well understood by those skilledin the art. In one aspect, the compounds of the present inventioninhibit the expression of IL 4R-α. The compounds of the invention canalso be used in the manufacture of a medicament for the treatment ofdiseases and disorders related to IL 4R-α expression.

Methods whereby bodily fluids, organs or tissues are contacted with aneffective amount of one or more of the antisense compounds orcompositions of the invention are also contemplated. Bodily fluids,organs or tissues can be contacted with one or more of the compounds ofthe invention resulting in modulation of IL 4R-α expression in the cellsof bodily fluids, organs or tissues. An effective amount can bedetermined by monitoring the modulatory effect of the antisense compoundor compounds or compositions on target nucleic acids or their productsby methods routine to the skilled artisan.

Thus, provided herein is the use of an isolated single- ordouble-stranded oligomeric compound targeted to IL 4R-α in themanufacture of a medicament for the treatment of a disease or disorderby means of the method described above. In a preferred embodiment, theoligomeric compound is a single stranded compound.

Salts, Prodrugs and Bioequivalents

The oligomeric compounds of the present invention comprise anypharmaceutically acceptable salts, esters, or salts of such esters, orany other functional chemical equivalent which, upon administration toan animal including a human, is capable of providing (directly orindirectly) the biologically active metabolite or residue thereof.Accordingly, for example, the disclosure is also drawn to prodrugs andpharmaceutically acceptable salts of the oligomeric compounds of thepresent invention, pharmaceutically acceptable salts of such prodrugs,and other bioequivalents.

The term “prodrug” indicates a therapeutic agent that is prepared in aninactive or less active form that is converted to an active form (i.e.,drug) within the body or cells thereof by the action of endogenousenzymes, chemicals, and/or conditions. In particular, prodrug versionsof the oligonucleotides of the invention are as SATE((S-acetyl-2-thioethyl) phosphate) derivatives according to the methodsdisclosed in WO 93/24510 or WO 94/26764. Prodrugs can also includeoligomeric compounds wherein one or both ends comprise nucleobases thatare cleaved (e.g., phosphodiester backbone linkages) to produce theactive compound.

The term “pharmaceutically acceptable salts” refers to physiologicallyand pharmaceutically acceptable salts of the compounds of the invention:i.e., salts that retain the desired biological activity of the parentcompound and do not impart undesired toxicological effects thereto.Sodium salts of antisense oligonucleotides are useful and are wellaccepted for therapeutic administration to humans. In anotherembodiment, sodium salts of dsRNA compounds are also provided.

Formulations

The oligomeric compounds of the invention may also be admixed,encapsulated, conjugated or otherwise associated with other molecules,molecule structures or mixtures of compounds.

The present invention also includes pharmaceutical compositions andformulations which include the antisense compounds of the invention. Thepharmaceutical compositions of the present invention may be administeredin a number of ways depending upon whether local or systemic treatmentis desired and upon the area to be treated. In a preferred embodiment,administration is topical to the surface of the respiratory tract,particularly pulmonary, e.g., by nebulization, inhalation, orinsufflation of powders or aerosols, by mouth and/or nose.

The pharmaceutical formulations of the present invention, which mayconveniently be presented in unit dosage form, may be prepared accordingto conventional techniques well known in the pharmaceutical industry.Such techniques include the step of bringing into association the activeingredients with the pharmaceutical carrier(s) or excipient(s). Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association the active ingredients with liquid carriers,finely divided solid carriers, or both, and then, if necessary, shapingthe product (e.g., into a specific particle size for delivery). In apreferred embodiment, the pharmaceutical formulations of the instantinvention are prepared for pulmonary administration in an appropriatesolvent, e.g., water or normal saline, possibly in a sterileformulation, with carriers or other agents to allow for the formation ofdroplets of the desired diameter for delivery using inhalers, nasaldelivery devices, nebulizers, and other devices for pulmonary delivery.Alternatively, the pharmaceutical formulations of the instant inventionmay be formulated as dry powders for use in dry powder inhalers.

A “pharmaceutical carrier” or “excipient” can be a pharmaceuticallyacceptable solvent, suspending agent or any other pharmacologicallyinert vehicle for delivering one or more nucleic acids to an animal andare known in the art. The excipient may be liquid or solid and isselected, with the planned manner of administration in mind, so as toprovide for the desired bulk, consistency, etc., when combined with anucleic acid and the other components of a given pharmaceuticalcomposition.

Combinations

Compositions of the invention can contain two or more oligomericcompounds. In another related embodiment, compositions of the presentinvention can contain one or more antisense compounds, particularlyoligonucleotides, targeted to a first nucleic acid and one or moreadditional antisense compounds targeted to a second nucleic acid target.Alternatively, compositions of the present invention can contain two ormore antisense compounds targeted to different regions of the samenucleic acid target. Two or more combined compounds may be used togetheror sequentially. Compositions of the instant invention can also becombined with other non-oligomeric compound therapeutic agents.

Nonlimiting Disclosure and Incorporation by Reference

While certain compounds, compositions and methods of the presentinvention have been described with specificity in accordance withcertain embodiments, the following examples serve only to illustrate thecompounds of the invention and are not intended to limit the same. Eachof the references, GenBank accession numbers, and the like recited inthe present application is incorporated herein by reference in itsentirety.

Example 1 Cell Types

The effect of oligomeric compounds on target nucleic acid expression wastested in the following cell types.

A549:

The human lung carcinoma cell line A549 was obtained from the AmericanType Culture Collection (Manassas, Va.). A549 cells were routinelycultured in DMEM, high glucose (Invitrogen Life Technologies, Carlsbad,Calif.) supplemented with 10% fetal bovine serum, 100 units per mlpenicillin, and 100 micrograms per ml streptomycin (Invitrogen LifeTechnologies, Carlsbad, Calif.). Cells were routinely passaged bytrypsinization and dilution when they reached approximately 90%confluence. Cells were seeded into 96-well plates (Falcon-Primaria#3872) at a density of approximately 5000 cells/well for use inoligomeric compound transfection experiments.

b.END:

The mouse brain endothelial cell line b.END was obtained from Dr. WernerRisau at the Max Plank Institute (Bad Nauheim, Germany). b.END cellswere routinely cultured in DMEM, high glucose (Invitrogen LifeTechnologies, Carlsbad, Calif.) supplemented with 10% fetal bovine serum(Invitrogen Life Technologies, Carlsbad, Calif.). Cells were routinelypassaged by trypsinization and dilution when they reached approximately90% confluence. Cells were seeded into 96-well plates (Falcon-Primaria#353872, BD Biosciences, Bedford, Mass.) at a density of approximately3000 cells/well for use in oligomeric compound transfection experiments.

Treatment with Oligomeric Compounds

When cells reach appropriate confluency, they are treated witholigonucleotide using a transfection lipid and method, such asLipofectin™ essentially by the manufacturer's instructions, asdescribed.

When cells reached 65-75% confluency, they were treated witholigonucleotide. Oligonucleotide was mixed with LIPOFECTIN™ InvitrogenLife Technologies, Carlsbad, Calif.) in Opti-MEM™-1 reduced serum medium(Invitrogen Life Technologies, Carlsbad, Calif.) to achieve the desiredconcentration of oligonucleotide and a LIPOFECTIN™ concentration of 2.5or 3 μg/mL per 100 nM oligonucleotide. This transfection mixture wasincubated at room temperature for approximately 0.5 hours. For cellsgrown in 96-well plates, wells were washed once with 100 μL OPTI-MEM™-1and then treated with 130 μL of the transfection mixture. Cells grown in24-well plates or other standard tissue culture plates are treatedsimilarly, using appropriate volumes of medium and oligonucleotide.Cells are treated and data are obtained in duplicate or triplicate.After approximately 4-7 hours of treatment at 37° C., the mediumcontaining the transfection mixture was replaced with fresh culturemedium. Cells were harvested 16-24 hours after oligonucleotidetreatment.

Other transfection reagents and methods (e.g., electroporation) fordelivery of oligonucleotides to the cell are well known. The method ofdelivery of oligonucleotide to the cells is not a limitation of theinstant invention.

Control Oligonucleotides

Control oligonucleotides are used to determine the optimal oligomericcompound concentration for a particular cell line. Furthermore, whenoligomeric compounds of the invention are tested in oligomeric compoundscreening experiments or phenotypic assays, control oligonucleotides aretested in parallel with compounds of the invention.

The concentration of oligonucleotide used varies from cell line to cellline. To determine the optimal oligonucleotide concentration for aparticular cell line, the cells are treated with a positive controloligonucleotide at a range of concentrations. The concentration ofpositive control oligonucleotide that results in 80% inhibition of thetarget mRNA is then utilized as the screening concentration for newoligonucleotides in subsequent experiments for that cell line. If 80%inhibition is not achieved, the lowest concentration of positive controloligonucleotide that results in 60% inhibition of the target mRNA isthen utilized as the oligonucleotide screening concentration insubsequent experiments for that cell line. If 60% inhibition is notachieved, that particular cell line is deemed as unsuitable foroligonucleotide transfection experiments. The concentrations ofantisense oligonucleotides used herein are from 50 nM to 300 nM when theantisense oligonucleotide is transfected using a liposome reagent and 1μM to 40 μM when the antisense oligonucleotide is transfected byelectroporation.

Example 2 Real-Time Quantitative PCR Analysis of IL 4R-α mRNA Levels

Quantitation of IL 4R-α mRNA levels was accomplished by real-timequantitative PCR using the ABI PRISM™ 7600, 7700, or 7900 SequenceDetection System (PE-Applied Biosystems, Foster City, Calif.) accordingto manufacturer's instructions.

Prior to quantitative PCR analysis, primer-probe sets specific to thetarget gene being measured were evaluated for their ability to be“multiplexed” with a GAPDH amplification reaction. After isolation theRNA is subjected to sequential reverse transcriptase (RT) reaction andreal-time PCR, both of which are performed in the same well. RT and PCRreagents were obtained from Invitrogen Life Technologies (Carlsbad,Calif.). RT, real-time PCR was carried out in the same by adding 20 μLPCR cocktail (2.5×PCR buffer minus MgCl₂, 6.6 nIM MgCl₂, 375 M each ofdATP, dCTP, dCTP and dGTP, 375 nM each of forward primer and reverseprimer, 125 nM of probe, 4 Units RNAse inhibitor, 1.25 Units PLATINUM®Taq, 5 Units MuLV reverse transcriptase, and 2.5×ROX dye) to 96-wellplates containing 30 μL total RNA solution (20-200 ng). The RT reactionwas carried out by incubation for 30 minutes at 48° C. Following a 10minute incubation at 95° C. to activate the PLATINUM® Taq, 40 cycles ofa two-step PCR protocol were carried out: 95° C. for 15 seconds(denaturation) followed by 60° C. for 1.5 minutes (annealing/extension).

Gene target quantities obtained by RT, real-time PCR were normalizedusing either the expression level of GAPDH, a gene whose expression isconstant, or by quantifying total RNA using RiboGreen™ (MolecularProbes, Inc. Eugene, Oreg.). GAPDH expression was quantified by RT,real-time PCR, by being run simultaneously with the target,multiplexing, or separately. Total RNA was quantified using RiboGreen™RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.).

170 μL of RiboGreen™ working reagent (RiboGreen™ reagent diluted 1:350in 10 mM Tris-HCl, 1 mM EDTA, pH 7.5) was pipetted into a 96-well platecontaining 30 μL purified cellular RNA. The plate was read in aCytoFluor 4000 (PE Applied Biosystems) with excitation at 485 nm andemission at 530 nm.

The GAPDH PCR probes have JOE covalently linked to the 5′ end and TAMRAor MGB covalently linked to the 3′ end, where JOE is the fluorescentreporter dye and TAMRA or MGB is the quencher dye. In some cell types,primers and probe designed to a GAPDH sequence from a different speciesare used to measure GAPDH expression. For example, a human GAPDH primerand probe set is used to measure GAPDH expression in monkey-derivedcells and cell lines.

Probes and primers for use in real-time PCR were designed to hybridizeto target-specific sequences. The primers and probes and the targetnucleic acid sequences to which they hybridize are presented in Table 2.The target-specific PCR probes have FAM covalently linked to the 5′ endand TAMRA or MGB covalently linked to the 3′ end, where FAM is thefluorescent dye and TAMRA or MGB is the quencher dye.

TABLE 2 Gene target-specific primers and probes for use in real-time PCRTarget Sequence Target Name Species SEQ ID NO Description Sequence(5′ to 3′) SEQ ID NO IL-4R alpha Human 1 Fwd Primer AATGGTCCCACCAATTGCA3 IL-4R alpha Human 1 Reverse Primer CTCCGTTGTTCTCAGGGATACAC 4 IL-4Ralpha Human 1 Probe TTTTTCTGCTCTCCGAAGCCC 5 IL-4R alpha Mouse 2 FwdPrimer TCCCATTTTGTCCACCGAATA 6 IL-4R alpha Mouse 2 Reverse PrimerGTTTCTAGGCCCAGCTTCCA 7 IL-4R alpha Mouse 2 ProbeTGTCACTCAAGGCTCTCAGCGGTCC 8

Example 3 Antisense Inhibition of Mouse IL-4R Alpha by OligomericCompounds

A series of oligomeric compounds was designed to target differentregions of mouse IL 4R-α. RNA, using published sequences cited inTable 1. The compounds are shown in Table 3. All compounds in Table 3are chimeric oligonucleotides (“gapmers”) 20 nucleotides in length,composed of a central “gap” region consisting of 10 2′-deoxynucleotides,which is flanked on both sides (5′ and 3′) by five-nucleotide “wings”.The wings are composed of 2′-O-(2-methoxyethyl) nucleotides, also knownas 2′-MOE nucleotides. The internucleoside (backbone) linkages arephosphorothioate throughout the oligonucleotide. All cytidine residuesare 5-methylcytidines. The compounds were analyzed for their effect ongene target mRNA levels by quantitative real-time PCR as described inother examples herein, using the target-specific primers and probesshown in Table 2. Data are averages from two experiments in which b.ENDcells were treated with 150 nM of the compounds in Table 3 usingLipofectin™. A reduction in expression is expressed as percentinhibition in Table 3. If the target expression level of oligomericcompound-treated cell was higher than control, percent inhibition isexpressed as zero inhibition. The target regions to which theseoligomeric compounds are inhibitory are herein referred to as “validatedtarget segments.”

TABLE 3 Inhibition of mouse IL 4R-α mRNA levels by chimericoligonucleotides having 2′-MOE wings and deoxy gap Target Target % ISIS# SEQ ID/GenBank Site Sequence (5′ to 3′) Inhib SEQ ID NO 231931Assm.fr. 1364 ACCCGCACAAGGTCCTGGGC 20  9 M64868.1/M64879.1 231932Assm.fr. 2204 CAGGTCTTACCATTACCACT 33 10 M64868.1/M64879.1 231933Assm.fr. 2506 GCCCACTCACTTCTGCAGGG 50 11 M64868.1/M64879.1 231934Assm.fr. 2804 CGGTTGTACCACGTGATGCT 51 12 M64868.1/M64879.1 231935Assm.fr. 2813 TGATACTCACGGTTGTACCA 29 13 M64868.1/M64879.1 231936Assm.fr. 3327 AGGAACTCACTTGGTAATGC  9 14 M64868.1/M64879.1 231937Assm.fr. 3559 TGTACCCTCTTACCTGTGCA 30 15 M64868.1/M64879.1 231929BB867141.1   49 CAAAAGGTGCCTGCGAGTTC 19 16 231930 BC012309.1  101GGCTGGGTTACAGGAACAAG  0 17 231928 M27959.1  900 AGCTGGAAGTGGTTGTACCA 2318 231860 2   78 AATCAGAAGCCAGGTCCCTC 66 19 231861 2  209CAAAAGGTGCCTGCACAAGG 34 20 231862 2  233 TGCAAAGCCGCCCCATTGGG 66 21231863 2  244 CAGGAACTTGGTGCAAAGCC 60 22 231864 2  330TAGTCAGAGAAGCAGGTGGG 44 23 231865 2  340 AGTGCGGATGTAGTCAGAGA 39 24231866 2  388 CTGAGAACTGCAGTCCACAG 58 25 231867 2  438GTGAGGTTTTCAGAGAACTC 28 26 231868 2  443 TGCATGTGAGGTTTTCAGAG 48 27231869 2  611 GTGTGAGGTTGTCTGGAGCT 63 28 231870 2  624ACATTGGTGTGGAGTGTGAG 38 29 231871 2  716 CTCTGGAGATGTTGACCATG 48 30231872 2  721 GTCCTCTCTGGAGATGTTGA 43 31 231873 2  726GGGTTGTCCTCTCTGGAGAT 32 32 231874 2  758 TGTAGGTCACATTATAGACT 66 33231875 2  891 GGGTTGTACCACGTGATGCT 27 34 231876 2  918TCACTCAGTCACAGATTTTC 70 35 231877 2 1014 AGCTGGAAGTCCATCTCCTG 23 36231878 2 1114 CTTCTTAATCTTGGTAATGC 40 37 231879 2 1121ACCATATCTTCTTAATCTTG 25 38 231880 2 1126 GTCCCACCATATCTTCTTAA 11 39231881 2 1131 ATCTGGTCCCACCATATCTT 36 40 231882 2 1136TGGGAATCTGGTCCCACCAT 43 41 231883 2 1225 GGTTGACTCCTGGCTTCGGG  7 42231884 2 1385 GGACGGTCCTGCTGACCTCC 65 43 231885 2 1390CCAGAGGACGGTCCTGCTGA 55 44 231886 2 1395 TCTGGCCAGAGGACGGTCCT 65 45231887 2 1424 TACAGCGCACCACACTGACA 80 46 231888 2 1430GCTCCATACAGCGCACCACA 72 47 231889 2 1435 AAACAGCTCCATACAGCGCA 77 48231890 2 1440 GCCTCAAACAGCTCCATACA 58 49 231891 2 1460CCTCCACATTCTGTACTGGG 75 50 231892 2 1505 CAGGTGACATGCTCAGGTCC 63 51231893 2 1510 GTTCTCAGGTGACATGCTCA 68 52 231894 2 1515CCGCTGTTCTCAGGTGACAT 88 53 231895 2 1575 AACAGGTTCTCAGTGAGCCG 62 54231896 2 1834 CCGGTAGGCAGGATTGTCTG 62 55 231897 2 1839AAACTCCGGTAGGCAGGATT 68 56 231898 2 1844 CACTAAAACTCCGGTAGGCA 71 57231899 2 1880 CCAGCTCTCCAGGATTTGGG 68 58 231900 2 1960TGGTGGCCCTGAAGAATGGG 30 59 231901 2 1991 GGATCTGCTCCCAGCTCTCC 85 60231902 2 1996 GTGAAGGATCTGCTCCCAGC 80 61 231903 2 2001CTCATGTGAAGGATCTGCTC 69 62 231904 2 2006 GGACACTCATGTGAAGGATC 52 63231905 2 2011 CTGCAGGACACTCATGTGAA 67 64 231906 2 2079TTCACTGCCTGCACAAACTC 60 65 231907 2 2084 CCTGCTTCACTGCCTGCACA 72 66231908 2 2166 CTGCTGAGCAGGCTCGAGAA 51 67 231909 2 2437GTCATCCCCAAAGGGCTTGG 69 68 231910 2 2442 CCCAGGTCATCCCCAAAGGG 71 69231911 2 2469 GTGAGGGACGAGTACACAAT 68 70 231912 2 2497TTGCTTCAGGTGGCCACACA 69 71 231913 2 2502 TGGTGTTGCTTCAGGTGGCC 71 72231914 2 2507 GGCTGTGGTGTTGCTTCAGG 51 73 231915 2 2719CTGGCTGGGAACAGGAGAGT 64 74 231916 2 2788 AGCAACAACAGCACACTCAC 78 75231917 2 2793 ACCTCAGCAACAACAGCACA 82 76 231918 2 2798CACAGACCTCAGCAACAACA 78 77 231919 2 2827 TCCCTGGCTTGGAGGAACCC 62 78231920 2 2859 CCTGCCAGCTGGGCTGTCTC 66 79 231921 2 2869TTCTGGGAAACCTGCCAGCT 73 80 231922 2 3340 ACTTTGGGCAATCAAGTTTG 32 81231923 2 3345 CAGTGACTTTGGGCAATCAA 64 82 231924 2 3350ACTGGCAGTGACTTTGGGCA 59 83 231925 2 3355 GGGTAACTGGCAGTGACTTT 56 84231926 2 3671 TAAAGACTTTATTGACATAA 41 85 231927 2 3678GACAAGATAAAGACTTTATT 41 86

All oligonucleotides targeted to the following regions of a GenBanksequence assembled from assembled from M64868.1 and M64879.1 wereeffective at inhibiting expression of IL 4R-α at least 40% as can bedetermined by the table above: nucleotides 2506-2525 and 2804-2323.These are validated target segments. All oligonucleotides targeted tothe following regions of SEQ ID NO: 2 were effective at inhibitingexpression of UL 4R-α at least 40% as can be determined by the tableabove: nucleotides 78-97; 233-263; 330-349; 388-407; 443-462; 611-630;716-740; 758-777; 918-9937; 1014-1033; 1114-1133; 1136-1155; 1385-1314;1424-1459; 1505-1534; 1575-1594; 1834-1863; 1880-1899; 1991-2030;2979-2103; 2166-2185; 2437-2461; 2469-2488; 2497-2526; 2719-2738;2788-2817; 2827-2846; 2859-2888; 3345-3374; and 3671-3697. These arevalidated target segments.

Example 4 Antisense Inhibition of Human IL 4R-α by Oligomeric Compounds

A series of oligomeric compounds was designed to target differentregions of human IL 4R-α RNA, using published sequences cited inTable 1. The compounds are shown in Tables 4 and 5. All compounds inTables 4 and 5 are chimeric oligonucleotides (“gapmers”) 20 nucleotidesin length, composed of a central “gap” region consisting of 102′-deoxynucleotides, which is flanked on both sides (5′ and 3′) byfive-nucleotide “wings”. The wings are composed of 2′-O-(2-methoxyethyl)nucleotides, also known as 2′-MOE nucleotides. The internucleoside(backbone) linkages are phosphorothioate throughout the oligonucleotide.All cytidine residues are 5-methylcytidines. The compounds were analyzedfor their effect on gene target mRNA levels by quantitative real-timePCR as described in other examples herein, using the humantarget-specific primers and probes shown in Table 2. Data are averagesfrom two experiments in which A549 cells were treated with 85 nM of thecompounds in Table 4, and 70 nM of the compound in Table 5, usingLipofectin™. A reduction in expression is expressed as percentinhibition in Tables 4 and 5. If the target expression level ofoligomeric compound-treated cell was higher than control, percentinhibition is expressed as zero inhibition. The target regions to whichthese oligomeric compounds are inhibitory are herein referred to as“validated target segments.”

TABLE 4 Inhibition of human IL 4R-α mRNA levels by chimericoligonucleotides having 2′-MOE wings and deoxy gap Target ISIS # SEQ IDNO Site Sequence (5′ to 3′) % Inhib SEQ ID NO 364941 1   21GTAAATCTTTAATTATCTGC  9  87 364945 1  234 CATGTTCCCAGAGCTTGCCA 19  88364946 1  246 CTGCAAGACCTTCATGTTCC 40  89 364947 1  287CAAGTAGAGATGCTCATGTA 30  90 364948 1  317 CAATTGGTGGGACCATTCAT 36  91364949 1  487 ACAGCAGCTGCTGCCCAGCC 51  92 364951 1  741AATCCCAGACTTCAGGGTGC 45  93 364952 1  777 GCACTGAGCCCAGGCCCTCA 56  94364953 1  917 CTGACATAGCACAACAGGCA 55  95 364954 1  931TAATCTTGGTGATGCTGACA 48  96 364955 1  936 TTTCTTAATCTTGGTGATGC 25  97364958 1 1160 CCCTGGAAAGGCATCTCTTT 54  98 364959 1 1175GCTGATTTTCCAGAGCCCTG 59  99 364960 1 1182 GCACCATGCTGATTTTCCAG 57 100364962 1 1492 CCCAGGGCATGTGAGCACTC 49 101 364963 1 1499AACTCATCCCAGGGCATGTG 59 102 364964 1 1509 TGCACTTGGGAACTCATCCC 49 103364965 1 1608 GCAAGTCAGGTTGTCTGGAC 54 104 364966 1 1708GTGGGTCTGGACCCAGCTCT 46 105 364967 1 1716 GGCCAGCAGTGGGTCTGGAC 48 106364968 1 1845 TGCCCCATGCTGGAGGACAT 37 107 364969 1 1976GAGAAGGCCTTGTAACCAGC 53 108 364970 1 2000 ACAGCACTGCTGGCAAGCAG 35 109364971 1 2038 CCCCACTGCTAGCCCCAAAC 24 110 364972 1 2043CTCTTCCCCACTGCTAGCCC 25 111 364973 1 2058 GAAAGGCTTATACCCCTCTT 62 112364974 1 2067 GAGGTCTTGGAAAGGCTTAT 55 113 364975 1 2082AGGGCAGCCAGGAATGAGGT 42 114 364976 1 2087 TCCCCAGGGCAGCCAGGAAT 37 115364977 1 2230 GCTTTGGCATGTCCTCTACC 50 116 364978 1 2301GGCTGAGTAGACAATGCCAC 26 117 364979 1 2315 AGGTGGCAGGTAAGGGCTGA 39 118364980 1 2390 CCACAGCAAGGACTGGCCAT 45 119 364981 1 2469CAGTGGAACCCCACCTGGAG 23 120 364983 1 2541 GAAGGATGATGAGGATTTAC 51 121364984 1 2548 CAGGATGGAAGGATGATGAG 41 122 364985 1 2569AGCTCTGAGCATTGCCAGGG 45 123 364986 1 2626 CCCTCATGTATGTGGGTCCC 48 124364987 1 2643 GACATGCACCTAAGAGACCC 49 125 364988 1 2674TAGTCCTCATCTGCAGACTC 50 126 364989 1 2731 AATCTGCCAGCCTGGCTGCC 41 127364991 1 2751 GGTTCTTCAAGTCTTTTGGA 56 128 364993 1 2772GGCCAATCACCTTCATACCA 47 129 364994 1 2836 GAGCCCAGCCCAATGCTGGG  7 130364995 1 2856 CTACTCTCATGGGATGTGGC 61 131 364996 1 2861GCCCTCTACTCTCATGGGAT 58 132 364997 1 2909 GGCCTCAGTTTTCCTGCAGG 30 133364998 1 2915 CCCAAGGGCCTCAGTTTTCC 55 134 364999 1 2952GAGGGAGCAGCCAACAACTC 31 135 365000 1 3048 AGACAGAGGCAGGTGGGCCT 35 136365001 1 3053 CAGTGAGACAGAGGCAGGTG 63 137 365002 1 3103CAAGTCATTCCCTTGATGGC 48 138 365004 1 3198 ATCAACCTAAGGAAGCTCTG 49 139365005 1 3238 TAACTGAACACCCCTTGACA  6 140 365006 1 3290AATTGTCCCTGCTTTAGTCA 16 141 365007 1 3297 GGCAGCAAATTGTCCCTGCT 55 142365008 1 3303 GTGTTTGGCAGCAAATTGTC 46 143 365009 1 3420GGGTAACTGGTGCCTTATGC 53 144 365010 1 3432 GGCCAACATGCAGGGTAACT 44 145365011 1 3477 ATTACTCAACCCAAGGTTCC 20 146 365012 1 3572AAGAAACTTTATTTATACAA  0 147 365013 1 3578 GAGACAAAGAAACTTTATTT  2 148365014 18636000- 8231 CCTAGAATTCAGTCTTCCCT 41 149 18639000 ofNM_010393.14 365015 18636000- 20215  GTTTCCATCTAGAGTACTAG 35 15018639000 of NT_010393.14 365016 18636000- 27651  GCCAAGGCACCTGCAGAGAG 38151 18639000 of NT_010393.14 365017 18636000- 47104 AGTGAGTGGCAGAGTCAGGA 48 152 18639000 of NT_010393.14 365018 18636000-49717  CTTCCAGTGTCTGCAAAAGC  0 153 18639000 of NT_010393.14

TABLE 5 Inhibition of human IL 4R-α mRNA levels by chimericoligonucleotides having 2′-MOE wings and deoxy gap Target ISIS # SEQ IDNO Site Sequence (5′ to 3′) % Inhib SEQ ID NO 364942 1  167AGCCACCCCATTGGGAGATG 88 154 364943 1  173 GAGCAAAGCCACCCCATTGG 83 155369517 1  176 CCAGAGCAAAGCCACCCCAT 51 156 369528 1  193TCACAGGGAACAGGAGCCCA 48 157 369529 1  194 CTCACAGGGAACAGGAGCCC 62 158369530 1  196 AGCTCACAGGGAACAGGAGC 44 159 369531 1  197CAGCTCACAGGGAACAGGAG 54 160 369532 1  199 GGCAGCTCACAGGGAACAGG 69 161369533 1  200 AGGCAGCTCACAGGGAACAG 64 162 369534 1  201CAGGCAGCTCACAGGGAACA 64 163 369535 1  202 CCAGGCAGCTCACAGGGAAC 58 164369536 1  203 ACCAGGCAGCTCACAGGGAA 65 165 369537 1  205GGACCAGGCAGCTCACAGGG 63 166 369538 1  206 AGGACCAGGCAGCTCACAGG 74 167369539 1  207 CAGGACCAGGCAGCTCACAG 66 168 369540 1  208GCAGGACCAGGCAGCTCACA 57 169 369541 1  209 AGCAGGACCAGGCAGCTCAC 48 170369542 1  210 CAGCAGGACCAGGCAGCTCA 46 171 369543 1  211GCAGCAGGACCAGGCAGCTC 48 172 369544 1  212 TGCAGCAGGACCAGGCAGCT 39 173369545 1  213 CTGCAGCAGGACCAGGCAGC 22 174 369546 1  215ACCTGCAGCAGGACCAGGCA 38 175 369547 1  217 CCACCTGCAGCAGGACCAGG 63 176369548 1  219 TGCCACCTGCAGCAGGACCA 57 177 369549 1  220TTGCCACCTGCAGCAGGACC 61 178 369550 1  221 CTTGCCACCTGCAGCAGGAC 60 179369551 1  222 GCTTGCCACCTGCAGCAGGA 44 180 369552 1  223AGCTTGCCACCTGCAGCAGG 42 181 364944 1  224 GAGCTTGCCACCTGCAGCAG 56 182369553 1  225 AGAGCTTGCCACCTGCAGCA 64 183 369554 1  226CAGAGCTTGCCACCTGCAGC 65 184 369555 1  227 CCAGAGCTTGCCACCTGCAG 66 185369556 1  228 CCCAGAGCTTGCCACCTGCA 70 186 369557 1  229TCCCAGAGCTTGCCACCTGC 50 187 369558 1  284 GTAGAGATGCTCATGTAGTC 50 188369559 1  353 AAAACCAGCTGGTACAACAG 40 189 369560 1  355GAAAAACCAGCTGGTACAAC 36 190 369561 1  428 TCCATGAGCAGGTGGCACAC 67 191369562 1  429 ATCCATGAGCAGGTGGCACA 71 192 369563 1  430CATCCATGAGCAGGTGGCAC 78 193 369564 1  431 TCATCCATGAGCAGGTGGCA 75 194369565 1  494 CCCTTCCACAGCAGCTGCTG 78 195 369566 1  496AGCCCTTCCACAGCAGCTGC 86 196 369567 1  497 GAGCCCTTCCACAGCAGCTG 71 197369568 1  499 AGGAGCCCTTCCACAGCAGC 74 198 369569 1  500AAGGAGCCCTTCCACAGCAG 76 199 369570 1  501 GAAGGAGCCCTTCCACAGCA 71 200369571 1  502 TGAAGGAGCCCTTCCACAGC 54 201 369572 1  503TTGAAGGAGCCCTTCCACAG 35 202 369573 1  504 CTTGAAGGAGCCCTTCCACA 51 203369574 1  506 GGCTTGAAGGAGCCCTTCCA 40 204 369575 1  508TGGGCTTGAAGGAGCCCTTC  0 205 369576 1  509 CTGGGCTTGAAGGAGCCCTT  0 206369577 1  510 GCTGGGCTTGAAGGAGCCCT  3 207 369578 1  530GCCCTGGGTTTCACATGCTC 64 208 369579 1  531 GGCCCTGGGTTTCACATGCT 62 209369580 1  619 TATACAGGTAATTGTCAGGG 53 210 369581 1  620TTATACAGGTAATTGTCAGG 55 211 369582 1  621 ATTATACAGGTAATTGTCAG 40 212369583 1  642 GTTGACTGCATAGGTGAGAT 70 213 369584 1  645AATGTTGACTGCATAGGTGA 72 214 369585 1  647 CAAATGTTGACTGCATAGGT 68 215369586 1  649 TCCAAATGTTGACTGCATAG 61 216 364950 1  735AGACTTCAGGGTGCTGGCTG 45 217 369587 1  736 CAGACTTCAGGGTGCTGGCT 63 218369588 1  737 CCAGACTTCAGGGTGCTGGC 63 219 369589 1  998TCCTGGATTATTATAGCCAC 45 220 364956 1  999 ATCCTGGATTATTATAGCCA 39 221369590 1 1000 CATCCTGGATTATTATAGCC 43 222 369591 1 1001GCATCCTGGATTATTATAGC 51 223 369592 1 1003 GAGCATCCTGGATTATTATA 45 224369593 1 1004 TGAGCATCCTGGATTATTAT 26 225 369594 1 1005CTGAGCATCCTGGATTATTA 52 226 369595 1 1006 CCTGAGCATCCTGGATTATT 41 227364957 1 1053 GCACTTGGCTGGTTCCTGGC 77 228 369596 1 1077GGTAAGACAATTCTTCCAGT 77 229 369597 1 1078 TGGTAAGACAATTCTTCCAG 57 230369598 1 1079 TTGGTAAGACAATTCTTCCA 66 231 369599 1 1080CTTGGTAAGACAATTCTTCC 73 232 369600 1 1082 AGCTTGGTAAGACAATTCTT 66 233369601 1 1083 GAGCTTGGTAAGACAATTCT 61 234 369602 1 1085AAGAGCTTGGTAAGACAATT 64 235 369603 1 1087 GCAAGAGCTTGGTAAGACAA 64 236369604 1 1088 GGCAAGAGCTTGGTAAGACA 76 237 369605 1 1090AGGGCAAGAGCTTGGTAAGA 44 238 369606 1 1092 ACAGGGCAAGAGCTTGGTAA 64 239369607 1 1093 AACAGGGCAAGAGCTTGGTA 69 240 369608 1 1094AAACAGGGCAAGAGCTTGGT 77 241 369609 1 1095 AAAACAGGGCAAGAGCTTGG 62 242369610 1 1096 GAAAACAGGGCAAGAGCTTG 54 243 369611 1 1098CAGAAAACAGGGCAAGAGCT 62 244 369612 1 1100 TCCAGAAAACAGGGCAAGAG 72 245369613 1 1184 GGGCACCATGCTGATTTTCC 71 246 369614 1 1221GCTCTCTGGCCAGAGGACTG 80 247 369615 1 1223 ATGCTCTCTGGCCAGAGGAC 68 248369616 1 1224 GATGCTCTCTGGCCAGAGGA 58 249 369617 1 1227GCTGATGCTCTCTGGCCAGA 64 250 369618 1 1395 GTCCAGGAACAGGCTCTCTG 76 251369619 1 1397 AGGTCCAGGAACAGGCTCTC 68 252 369620 1 1398CAGGTCCAGGAACAGGCTCT 43 253 369621 1 1399 GCAGGTCCAGGAACAGGCTC 59 254369622 1 1400 AGCAGGTCCAGGAACAGGCT 45 255 364961 1 1401GAGCAGGTCCAGGAACAGGC 54 256 369623 1 1506 ACTTGGGAACTCATCCCAGG 58 257369624 1 1507 CACTTGGGAACTCATCCCAG 58 258 369625 1 1508GCACTTGGGAACTCATCCCA 66 259 369626 1 1670 CTCAGGGAGTTGCTGAAGCT 63 260369627 1 1671 GCTCAGGGAGTTGCTGAAGC 62 261 369628 1 1673TGGCTCAGGGAGTTGCTGAA 28 262 369629 1 1674 CTGGCTCAGGGAGTTGCTGA 47 263369630 1 1676 GACTGGCTCAGGGAGTTGCT 65 264 369631 1 1700GGACCCAGCTCTCTGGGACA 57 265 369632 1 1701 TGGACCCAGCTCTCTGGGAC 61 266369633 1 1703 TCTGGACCCAGCTCTCTGGG 46 267 369634 1 1705GGTCTGGACCCAGCTCTCTG 70 268 369635 1 1706 GGGTCTGGACCCAGCTCTCT 65 269369636 1 1777 TGGTTGGCTCAGAGAGCTGG 63 270 369637 1 1779AGTGGTTGGCTCAGAGAGCT 51 271 369638 1 1780 CAGTGGTTGGCTCAGAGAGC 64 272369639 1 1781 ACAGTGGTTGGCTCAGAGAG 57 273 369640 1 1782CACAGTGGTTGGCTCAGAGA 71 274 369641 1 1997 GCACTGCTGGCAAGCAGGCT 52 275369642 1 2056 AAGGCTTATACCCCTCTTCC 81 276 369643 1 2057AAAGGCTTATACCCCTCTTC 82 277 364973 1 2058 GAAAGGCTTATACCCCTCTT 60 112369644 1 2059 GGAAAGGCTTATACCCCTCT 80 279 369645 1 2060TGGAAAGGCTTATACCCCTC 84 280 369646 1 2062 CTTGGAAAGGCTTATACCCC 68 281369647 1 2064 GTCTTGGAAAGGCTTATACC 59 282 369648 1 2065GGTCTTGGAAAGGCTTATAC 58 283 369649 1 2066 AGGTCTTGGAAAGGCTTATA 77 284364974 1 2067 GAGGTCTTGGAAAGGCTTAT 60 113 369650 1 2068TGAGGTCTTGGAAAGGCTTA 59 286 369651 1 2126 AGTCCAAAGGTGAACAAGGG 50 287369652 1 2128 CCAGTCCAAAGGTGAACAAG 55 288 369653 1 2130GTCCAGTCCAAAGGTGAACA 50 289 369654 1 2131 TGTCCAGTCCAAAGGTGAAC 52 290369655 1 2403 TCCACAGCAGCAGCCACAGC 57 291 369656 1 2424TACTCTTCTCTGAGATGCCC 86 292 369657 1 2526 TTTACTCTTCTCTGAGATGC 71 293369658 1 2528 GATTTACTCTTCTCTGAGAT 57 294 369659 1 2529GGATTTACTCTTCTCTGAGA 67 295 364982 1 2530 AGGATTTACTCTTCTCTGAG 68 296369660 1 2531 GAGGATTTACTCTTCTCTGA 87 297 369661 1 2532TGAGGATTTACTCTTCTCTG 83 298 369662 1 2578 TCTGGCTTGAGCTCTGAGCA 69 299369663 1 2579 GTCTGGCTTGAGCTCTGAGC 68 300 364990 1 2743AAGTCTTTTGGAAATCTGCC 69 301 364992 1 2763 CCTTCATACCATGGTTCTTC 81 302365003 1 3168 GAGCACCTCTAGGCAATGAC 82 303

Oligonucleotides targeted to the following nucleotides of SEQ ID NO: 3were effective at inhibiting the expression of human IL 4R-α at leastabout 40% as can be determined by the tables above: nucleotides 167-265;284-303; 353-372; 428-450; 487-525; 530-550; 619-640; 642-668; 735-760;777-796; 917-950; 998-1025; 1053-1072; 1077-1121; 1160-1203; 1221-1246;1395-1420; 1492-1528; 1608-1627; 1670-1695; 1700-1735; 1777-1801;1976-1995; 1997-2016; 2056-2088, 2056-2101; 2126-2150; 2230-2349;2390-2422; 2524-2598; 2626-2662; 2674-2693; 2731-2791; 2856-2880;2915-2934; 3053-3072; 3103-3122; 3168-3187; 3198-3217; 3297-3322; and3420-3451. These are validated target segments. Although someoligonucleotides within each nucleotide region did not inhibitexpression at least 40%, they substantially overlapped (i.e., at least80% overlapped) oligonucleotides effective at inhibiting expression atleast 40%. All oligonucleotides targeted to the following regions of SEQID NO: 1 were effective at inhibiting expression of IL 4R-α at least 50%as can be determined by the tables above: nucleotides 284-303; 428-450;494-525; 530-550; 642-668; 1053-1072; 1184-1203; 1221-1246; 1506-1527;1777-1801; 1976-2016; 2056-2101; 2126-2150; 2230-2349; 2403-2422;2524-2551; 2578-2598; 2743-2782; 2856-2880; 2915-2934 and 3168-3187.These are validated target segments. All oligonucleotides targeted tothe following regions of GenBank nucleotides 18636000-18639000 ofNT_(—)010393.14 were effective at inhibiting expression of IL 4R-α atleast 40% as can be determined by the table above: nucleotides 8231-8250and 47104-47123. These are validated target segments.

Example 5 Screening of Oligonucleotides Containing NucleotideMismatches, Dose Response

Based on the screening above, ISIS 231894 was selected for furtherstudy. A series of oligonucleotides were designed based on ISIS 231894containing 1, 3, 5, and 7 mismatch nucleobases as shown in Table 6below. It should be noted that the mismatches are interspersedthroughout the central portion of the compounds, rather than at theends. This decreases the affinity of the oligonucleotide for the targetmRNA more than mismatch oligonucleotides at the ends. Such concepts arewell known and understood by those skilled in the art. Theoligonucleotides are 5-10-5 MOE-gapmers, as is ISIS 231894. All cytidineresidues are 5-methylcytidines. The mismatch bases are underlined.

TABLE 6 Oligonucleotides targeted to mouse IL 4R-α containing mismatchesTarget # mis- SEQ ISIS # SEQ ID NO match Sequence (5′ to 3′) ID NO231894 2 0 CCGCTGTTCTCAGGTGACAT  53 352489 2 1 CCGCTGTTCTCAGGTGACAT  53352490 2 3 CCGCTGATCACAGCTGACAT 304 352491 2 5 CCGCTCATCACTGCTGACAT 305352492 2 7 CCACTCATCACTGCTGACTT 306

The compounds were analyzed for their effect on gene target mRNA levelsby quantitative real-time PCR as described in other examples hereinusing the target specific primers shown in Table 2. Data are averagesfrom two experiments in which b.END cells were treated with theconcentrations of the compounds listed.

TABLE 7 Inhibition of mouse IL 4R-α by chimeric, mismatcholigonucleotides SEQ Number 25 50 100 ID Isis No mismatch 1 nM 5 nM 10nM nM nM nM NO 231894 Parent 100 54 43 31 21 21 53 352489 1 mm 74 55 5251 44 49 53 352490 3 mm 92 106 98 88 89 88 304 352491 5 mm 104 104 97102 114 90 305 352492 7 mm 109 118 121 104 88 69 306

Oligonucleotides having at least three mismatched bases interspersedwithin the central portion of the compound were not able to reduce theexpression of the target RNA by at least 40% even at the highest dosesof oligonucleotide tested.

Example 6 Mouse Models of Allergic Inflammation

Asthma is a complex disease with variations on disease severity andduration. In view of this, multiple animal models have been designed toreflect various aspects of the disease (see FIG. 1). It is understoodthat the models have some flexibility in regard to days of sensitizationand treatment, and that the timelines provided reflect the days usedherein. There are several important features common to human asthma andthe mouse model of allergic inflammation. One of these is pulmonaryinflammation, in which production of Th2 cytokines, e.g., IL 4, IL 5, IL9, and IL 13 is dominant. Another is goblet cell metaplasia withincreased mucus production. Lastly, airway hyperresponsiveness (AHR)occurs, resulting in increased sensitivity to cholinergic receptoragonists such as acetylcholine or methacholine.

Ovalbumin Induced Allergic Inflammation—Acute Model

The acute model of induced allergic inflammation is a prophylaxistreatment paradigm. Animals are sensitized to allergen by systemicadministration (i.e., intraperitoneal injection), and treated with thetherapeutic agent prior to administration of the pulmonary allergenchallenge (see FIG. 1A). In this model, there is essentially nopulmonary inflammation prior to administration of the therapeutic agent.

Balb/c mice (Charles River Laboratory, Taconic Farms, N.Y.) weremaintained in micro-isolator cages housed in a specific pathogen free(SPF) facility. The sentinel cages within the animal colony surveyednegative for viral antibodies and the presence of known mouse pathogens.Mice were sensitized and challenged with aerosolized chicken OVA.Briefly, 20 ug of alum precipitated OVA was injected intraperitoneallyon days 0 and 14. On days 24, 25 and 26, the animals were exposed for 20minutes to 1% OVA (in saline) by ultrasonic nebulization. On days 17,19, 21, 24 and 26 animals were dosed with 1 ug/kg or 10 ug/kg of ISIS231894 or the mismatch control oligonucleotide using an aerosol deliverysystem. Oligonucleotides were suspended in 0.9% sodium chloride anddelivered via inhalation using a nose-only exposure system. A Lovelacenebulizer (Model 01-100) was used to deliver the oligonucleotide into anair flow rate of 1.0 liter per minute feeding into a total flow rate of10 liters per minute. The exposure chamber was equilibrated with anoligonucleotide aerosol solution for 5 minutes before mice were placedin a restraint tubes attached to the chamber. Restrained mice weretreated for a total of 10 minutes. Analysis was performed on day 28.

Airway Hyperreponsiveness in Response to Methacholine

Airway responsiveness was assessed by inducing airflow obstruction witha methacholine aerosol using a noninvasive method. This method usedunrestrained conscious mice that are placed into a test chamber of aplethsmograph (Buxco Electronics, Inc. Troy, N.Y.). Pressure differencebetween this chamber and a reference chamber were used extrapolateminute volume, breathing frequency and enhanced pause (Penh). Penh is adimensionless parameter that is a function of total pulmonary airflow inmice (i.e. the sum of the airflow in the upper and lower respiratorytracts) during the respiratory cycle of the animal. The lower the Penh,the greater the airflow. This parameter is known to closely correlatewith lung resistance as measured by traditional, invasive techniquesusing ventilated animals as shown below (see also Hamelmann et al.,1997).

ISIS 231894, but not the mismatch control oligonucleotide, caused asignificant (p≦0.05 for both 1 ug/kg and 10 ug/kg vs. vehicle treatedcontrols) dose dependent suppression in methacholine induced AHR insensitized mice as measured by whole body plethysmography.

Airway hyperresponsiveness to methacholine challenge was also evaluatedusing an invasive technique designed to monitor changes in airwayresistance and pulmonary compliance. Mice were weighed and anesthetizedwith ketamine (150 mg/kg) mixed with xylazine (10 mg/kg). A trachestomywas performed and the mice were ventilated using the Flexivent system(SCIREQ, Montreal, Canada) using traditional mouse parameters (Adler, Aet al. J Appl Physiol 97: 286-292, 2004). Increasing concentrations ofmethacholine were aerosolized using the Flexivent system with an Aeroneblab nebulizer system, and resistance (RL) and compliance (CL) weremeasured.

ISIS 231894, but not the mismatch control oligonucleotide, reducedairway resistance (p<0.05 for 100 ug/kg vs. vehicle treated controls)and increased lung compliance (p<0.05 for 100 ug/kg vs. vehicle treatedcontrols) compared to measurements performed in control animals thatinhaled saline only.

Data are presented as group means +/−SEM, N=4-6/group. * p<0.05 vs.vehicle treated controls by Student's T-test.

TABLE 8 Measurement of AHR by Flexivent in response to methacholine inthe acute mouse OVA model Resistance (cm H2O-S/mL) Compliance (mL/cmH2O)Naïve  1.54 +/− 0.21*  0.029 +/− 0.005* Vehicle 4.51 +/− 1.12 0.010 +/−0.003 231894  1.70 +/− 0.34*  0.030 +/− 0.006* 352492 3.40 +/− 0.650.014 +/− 0.004

These data confirm that oligonucleotides targeted to IL 4R-α iseffective in the treatment of AHR in a prophylaxis paradigm.

Inflammatory Cell Infiltration

The effect of ISIS 231894 on inflammatory cell profiles was analyzed.Cell differentials were performed on bronchial alveolar lavage (BAL)fluid collected from lungs of the treated mice after injection of alethal dose of ketamine. Treatment with ISIS 231894, but not themismatch control, resulted in a significant (p≦0.05 for both 1 ug/kg and10 ug/kg vs. vehicle treated controls) decrease in BAL eosinophilinfiltration. These results demonstrate that an oligonucleotide targetedto IL4R-α decreased pulmonary inflammation by decreasing eosinophilinfiltration.

A second experiment was performed to confirm the efficacy of ISIS 231894to decrease AHR and eosinophilia in the acute model. Mice were dosedwith OVA as described above. On days 17, 19, 21, 24 and 26, mice weredosed with 10 ug/kg ISIS 231894, mismatch control oligonucleotide, orvehicle (i.e. saline). ISIS 231894, but not the mismatch controloligonucleotide significantly reduced AHR and eosinophilia as comparedto treatment with mismatch oligonucleotide or vehicle alone.

These data demonstrate that IL 4R-α is a valid target for the preventionof AHR and lung inflammation, and diseases associated therewith.

Mouse Model of Allergic Inflammation—Rechallenge Model

The rechallenge model of induced allergic inflammation allows testing ofa pharmacologic approach in mice that have been previously sensitizedand then exposed to an aeroallergen. During the first set of localallergen challenges, he mice develop allergen-specific memory Tlymphocytes. Subsequent exposure to a second set of inhaled allergenchallenges produces an enhanced inflammatory response in the lung, asdemonstrated by increased levels of Th2 cytokines in lavage fluid. Therechallenge model of allergic inflammation includes a second series ofaerosolized administration of OVA on days 59 and 60 in addition to thetwo IP OVA administrations on days 0 and 14 and the nebulized OVAadministration of days 24, 25 and 26 of the acute model (see FIG. 1B).Using this model, oligonucleotide treatment occurs after the first setof local allergen challenges. This also allows for the evaluation of thetarget's role in a recall response, as opposed to an initial immuneresponse.

In the rechallenge model, mice were treated with 10, 100 or 500 ug/kg ofeither ISIS 231894 or a 7 basepair mismatch control oligonucleotide(ISIS 352492) on days 52, 54, 56, 59 and 61 delivered by nose onlyinhalation. The study endpoints included many of those used in the acutemodel: Penh response (i.e., A-HR reduction), inflammatory cells in BAL,mucus accumulation, and lung histology. IL 4R-αprotein reduction inpulmonary structural and inflammatory cells was also evaluated.

Inflammatory IL4R-α Expression Profile

Lungs were harvested 6 hours following the second nebulized OVAre-challenge on Day 67. Lung cells were recovered after collagenasetreatment of the tissue and analyzed by flow cytometry. IL 4R-α proteinexpression was measured on the surface of a mixed population of lungeosinophils and macrophages (CD11b-positive, GR-1 negative or low);CD11c-positive and MHC class II-positive dendritic cells; andE-cadherin-positive epithelial cells. Data are expressed as meanfluorescence intensity +/−SEM, N=4/group. *p<0.05 vs. vehicle treatedcontrols by Student's T-test.

TABLE 9 IL 4Rα Cell Surface Protein Immunostaining (Mean FluorescenceIntensity) on Inflammatory and Epithelial Cells recovered from Lungs ofAllergen Re-challenged Mice Eos/Macs Epithelial Cells Treatments (MFI)DC (MFI) (MFI) Naive 142 +/− 27 184 +/− 40  63 +/− 22 Vehicle 181 +/− 26181 +/− 25 181 +/− 15 231894  10 ug/kg 120* +/− 7.8  200 +/− 38 100 +/−42 100 126* +/− 14  171 +/− 29 96* +/− 18 500 113* +/− 9.9  124* +/− 20 120 +/− 64 352492  10 ug/kg 135 +/− 32 206 +/− 26 144 +/− 30 100 172 +/−23 190 +/− 15  139 +/− 8.1 500 186 +/− 32 257 +/− 87 149 +/− 24

These data demonstrate that oligonucleotides targeted to IL 4R-α iseffective at reducing cell surface expression of IL 4R-α on multiplepopulations of cells within the lung.

Airway Hyperreponsiveness in Response to Methacholine as Determined byPenh

On day 60, AHR was analyzed by Penh as described above. A significantreduction in methacholine induced AHR was observed in animals inhaling1.0 or 10.0 ug/kg ISIS 231894, but not 10 ug/kg of the mismatch controloligonucleotide (ISIS 352492), as compared to vehicle control animals ascan be seen in Table 10 below. Data are presented as group means,N=10/group. * p<0.05 vs. vehicle treated controls by Student's T-test.

TABLE 10 Measurement of AHR by Penh in response to methacholine in arechallenge mouse model Methacholine dose (mg/kg) Treatments Baseline 03 6 12 25 50 100 Naïve 0.53 0.48 0.57 1.10 2.10 2.70 2.61 2.86* Vehicle0.60 0.52 0.97 1.52 2.16 3.16 4.46 5.13 231894 10 ug/kg 0.56 0.51 0.921.33 1.98 2.37 2.88 3.38* 231894 1.0 ug/kg 0.60 0.55 0.94 1.34 1.88 2.283.12 3.48* 231894 0.1 ug/kg 0.55 0.51 0.88 1.40 2.12 2.54 3.40 4.06231894 0.01 ug/kg 0.54 0.53 0.85 1.37 2.15 3.18 4.12 4.85 231894 0.001ug/kg 0.54 0.48 0.88 1.30 2.25 3.03 4.9 5.76 352492 10 ug/kg 0.57 0.520.93 1.36 2.24 3.17 3.75 4.14

These results demonstrate that oligonucleotides targeted to IL4R-α areeffective in the suppression of AHR.

Cytokine and Chemokine Expression in Bronchiolar Lavage Fluid

Pulmonary inflammation was also monitored through quantitation of Th2cytokines and chemokines, and eosinophils in the airways. The productionof Th2 cytokines and chemokines in the lung following aeroallergenexposure is associated with the induction of pulmonary inflammation andairway hyperresponsiveness. BAL fluid was collected, and the level ofTh2 cytokines and chemokines were quantitated by ELISA on Day 67, 6hours after the second nebulized OVA challenge in mice (n=4/group). Thelevel of IL-13 was significantly (p<0.05) decreased at all three dosesof Isis 231894. KC, the mouse analog of human IL-8, was significantly(p<0.05) decreased at the two higher doses of Isis 231894, and IL-5 andMCP-1 were significantly (p<0.05 vs. vehicle treated controls) decreasedat the 500 ug/kg dose. Cytokine concentrations were determined fromlinear regression analysis of multi-point standard curves. The mismatcholigonucleotide, Isis 352942, had no effect on Th2 cytokine levels.These data demonstrate that inhibition of IL-4Rα expression is effectivein decreasing Th2 cytokine and chemokine expression, specifically Th2cytokine expression following allergen challenge, which is related topulmonary inflammation and airway hyperresponsiveness.

Inflammatory Cell Infiltration

Cell differentials were performed on the BALF. The percent ofeosinophils in BAL fluid was significantly reduced as compared to BALFfrom vehicle treated control animals. Results are shown in Table 12.Data are presented as group means, N=10/group. * p<0.05 vs. vehicletreated controls by Student's T-test as compared to vehicle control.

TABLE 11 Measurement of Airway Inflammatory Cells in AllergenRe-challenged Mice Treatments Mac Lym Eos Neu Naïve 97.0 0.9  0.5* 1.6Vehicle 31.0 5.7 59.9  3.4 231894  10 ug/kg 72.1 4.2 23.0* 0.7 2318941.0 ug/kg 69.8 5.1 24*   0.6 231894 0.1 ug/kg 53.1 5.6 38*   3.3 2318940.01 ug/kg  47.3 4.8 44.1* 3.8 231894 0.001 ug/kg  38.9 4.6 53.2  3.3

These data demonstrate that oligonucleotides targeted to IL4R-α areeffective at decreasing eosinophilia in the lung in response to allergenchallenge.

Mucus Production

Mucus is an indicator of pulmonary inflammation. Muc5AC gene expression,mucus levels, and goblet cell metaplasia in the airways of OVAre-challenged mice were analyzed. Muc5AC mRNA levels were analyzed byquantitative RT-PCR in extracts from lung tissue harvested on Day 69.Expression levels were normalized to G3PDH expression. Muc 5AC/β-actinmRNA ratio significantly (p<0.05 vs. the vehicle treated control group)decreased with 231894 as compared to vehicle treatment. No decrease wasobserved in 352492 treated animals (n=4). There was also a significant(p<0.05 vs. the vehicle treated control group) decrease in mucus asdetermined by digital imaging of PAS-stained lungs from mice followingtreatment with IL 4R-α ASO, but not with saline or mismatch controloligonucleotide.

These data further demonstrate that an IL4R-α targeted antisenseoligonucleotide approach is efficacious in the presence of establishedimmunological memory, and that IL4R-α is an appropriate target for theamelioration, and/or treatment of AHR and pulmonary inflammation, anddiseases associated therewith.

Mouse model of allergic inflammation—chronic model

The chronic model of induced allergic inflammation uses a therapeutictreatment regimen, with ASO treatment initiated after the establishmentlocal pulmonary inflammation. The chronic model recapitulates some ofthe histological features of severe asthma in humans, including collagendeposition and lung tissue remodeling. The chronic OVA model produces amore severe disease than that observed in the acute of rechallengedmodel.

This model includes intranasal OVA administration on days 27-29, 47, 61,and 73-75, at a higher dose (500 ug) than in the acute and chronicmodels, in addition to the two OVA IP administrations on days 0 and 14(see FIG. 1C). Intranasal administration of the allergen results in ahigher dose of the allergen delivered to the lungs relative to deliveryby nebulizer. The increased number of allergen challenges produces moresevere inflammatory events, resulting in increased lung damage andpathology more reflective of clinical asthma than other models, in theabsence of therapeutic interventions. Endpoints tested were similar tothose in the acute and rechallenge model, including Penh (AHR), BALinflammatory cells and cytokines and mucus accumulation. This model alsoallows for the analysis of endpoints typically associated with chronicdiseases, such as asthma and COPD, including subepithelial fibrosis,collagen deposition, enhanced goblet cell metaplasia, and smooth musclecell hyperplasia.

Oligonucleotide, either ISIS 231894 or ISIS 352942 was administered bynose-only aerosol at a dose of either 5 ug/kg or 500 ug/kg on days 31,38, 45, 52, 59, 66 and 73. Analysis of endpoints was performed on day76, except cytokines which were evaluated on day 62, 6 hours post OVAchallenge.

Airway Hyperreponsiveness in Response to Methacholine as Determined byPenh

Treatment of mice with both doses of ISIS 231894 resulted in asignificant decrease in methacholine induced AHR as compared totreatment with vehicle (i.e. saline). Data are presented as group means,N=10/group. * p<0.05 vs. vehicle treated controls by Student's T-test.

TABLE 12 Measurement of AHR by Penh in response to methacholine in achronic allergic inflammation model Methacholine concentration (mg/ml)Treatment Baseline 0 3 6 12 25 50 100 Naïve 0.55 0.54 0.83 1.34 1.782.20 2.49 2.98* Vehicle 0.67 0.59 1.38 2.01 2.95 5.00 5.86 6.17 231894500 ug/kg 0.68 0.66 1.48 1.77 2.29 3.03 3.79 4.50* 231894  5 ug/kg 0.740.73 1.50 1.96 2.05 2.68 4.13 4.41*

These data demonstrate that oligonucleotides targeted to IL4R-αdelivered using at therapeutic treatment regimen are effective atpreventing AHR in response to methacholine challenge.

Inflammatory Cell Infiltration

Treatment of mice with the higher dose of 231894 resulted in asignificant decrease in the percent of eosinophils in BAL fluid ascompared to vehicle control. Both doses of ISIS 231894 significantlyreduced the percent neutrophils in BAL as compared to vehicle control.Data are presented as group means, N=7/group. * p<0.05 vs. vehicletreated controls by Student's T-test as compared to vehicle control.

TABLE 13 Measurement of inflammatory cell infiltration in a chronicallergic inflammation mouse model Day 62 Day 76 Treatments Mac Lym EosNeu Mac Lym Eos Neu Naïve 91.8 1.6 0.2* 6.4* 91.3 2.2 1.2* 5.3* Vehicle25.4 6.4 12.2 56.0 37.5 3.4 48.5 10.6 231894 500 58.2 2.4 4.8* 34.6*61.9 4.8 29.7* 3.6* ug/kg 231894  5 50.7 6.8 4.5* 38.0* 42.8 6.4 44.56.3 ug/kg

These data demonstrate that oligonucleotides targeted to IL4R-αdelivered using a therapeutic treatment regimen are effective atdecreasing eosinophilia and neutrophilia in the lung in response toallergen challenge.

Cytokine Expression in Bronchiolar Lavage Fluid

Pulmonary inflammation was also monitored by cytokine and chemokineexpression and inflammatory cell infiltration. BALF was collected, andthe level of four Th2 cytokines were quantitated by ELISA on day 62, 6hours post allergen challenge. Analysis of BAL fluid revealed asignificant reduction in IL-5 and KC in high dose 231894 treated animalsas compared to vehicle treated animals.

These data further confirm the utility of oligonucleotides targeted toIL 4R-α for the amelioration and treatment of AHR and pulmonaryinflammation and diseases associated therewith.

Example 7 Mouse Model of Allergic Inflammation, Analysis for NasalRhinitis Endpoints

Mouse models of allergen-induced acute and chronic nasal inflammationsimilar to those above have been used to study allergic rhinitis in mice(Hussain et al., Larangyoscope. 112: 1819-1826. 2002; Iwasaki et al., J.Allergy Clin Immunol. 112: 134-140. 2003; Malm-Erjefaelt et al., Am JRespir Cell Mol. Biol. 24:352-352. 2001; McCusker et al., J Allergy ClinImmunol., 110: 891-898; Saito et el., Immunology. 104:226-234. 2001). Inall of the models, the mice were sensitized to OVA by injection, asabove, followed by intranasal OVA instillation.

The most substantial difference in the models is in the endpointsanalyzed. Endpoints include, but are not limited to, the amount ofsneezing and nasal scratching immediately after administration ofallergen challenge (i.e. intranasal OVA), and nasal histology includingmucus and eosinophil counts and measurements of cytokines or otherinflammatory products in nasal lavage fluid or nasal tissues. Methodsfor performing such analyses are detailed in the references cited whichare incorporated herein by reference. Administration of oligonucleotidestargeted to IL-4R alpha decrease nasal inflammation, as evidenced byfewer infiltrating eosinophils quantitated by digital imaging, and fewernasal rubs and sneezes per unit of time in IL 4R-α ASO treated animalsas compared to saline treatment.

Example 8 Rodent Model of Smoking Induced Pulmonary Disease

Smoking is known to cause lung irritation and inflammation which canresult in a number of diseases in humans including, but not limited to,emphysema and COPD. A number of smoking animal models are well known tothose skilled in the art including those utilizing mice (Churg et al.,2002. Am. J. Respir. Cell. Mol. Biol. 27:368-347; Churg-et al., 2004.Am. J. Respir. Crit. Care Med. 170:492-498, both incorporated herein byreference), rats (e.g., Sekhon et al., 1994. Am. J. Physiol.267:L557-L563, incorporated herein by reference), and guinea pigs(Selman et al., 1996. Am J. Physiol. 271:L734-L739, incorporated hereinby reference). Animals are exposed to whole smoke using a smokingapparatus (e.g., Sekhon et al., 1994. Am. J. Physiol. 267:L557-L563)well known to those skilled in the art.

Changes in lung physiology are correlated with dose and time ofexposure. In short term studies, cell proliferation and inflammationwere observed. In one study, exposure of rats to 7 cigarettes for 1, 2,or 7 days resulted in proliferation of pulmonary artery walls at thelevel of the membranous bronchioles (MB), respiratory bronchioles (RB),and alveolar ducts (AD). Endothelial cell proliferation was only presentin vessels associated with AD. In a separate study (Churg et al., 2002.Am. J. Respir. Cell. Mol. Biol. 27:368-347), mice exposed to whole smokefrom four cigarettes were shown to have an increase in neutrophils,desmosine (an indicator of elastin breakdown), and hydroxyproline (anindicator of collagen breakdown) after only 24 hours. In a long termstudy, an emphysema-like state was induced (Churg et al., 2004. Am. J.Respir. Crit. Care Med. 170:492-498). Mice exposed to whole smoke fromfour cigarettes using a standard smoking apparatus, for five days perweek for six months were found to have an increase in neutrophils andmacrophages in BALF as compared to control mice. Whole lung matrixmetalloproteinases (MMP)-2, -9, -12, and -13, and matrix type-1 (MT-1)proteins were increased. An increase in matrix breakdown products wasalso observed in BALF. These markers correlate with tissue destructionand are observed in human lungs with emphysema.

These models can be used to determine the efficacy of therapeuticinterventions for the prevention, amelioration, and/or treatment of thedamage and disease caused by cigarette smoke and/or other insults.Administration of oligonucleotide can be performed prior to, concurrentwith, and/or after exposure to smoke to provide a prophylactic ortherapeutic model. ISIS 231894 is 100% complimentary to both mouse andrat IL 4R-α; therefore, it can be used in both mouse and rat studies.Dose ranges are determined by the time of oligonucleotide administrationrelative to smoke inhalation, with lower doses (e.g., 1-100 ug/kg)required for prevention of lung damage. Higher doses (e.g., 100-1000ug/kg) are required for treatment after, or alternating with, smokeexposure. Positive control (e.g., smoke exposure, no oligonucleotideadministration) and negative control (e.g., no smoke exposure, with orwithout oligonucleotide treatment) animals are also analyzed.

Endpoints for analysis include those discussed in the asthma modelsabove. Functional endpoints include A-HR, resistance and compliance.Morphological changes include BAL cell, cytokine levels, histologicaldeterminations of alveolar destruction (i.e., increase in alveolarspace) and airway mucus accumulation, as well as tissue markers ofdisease including collagen and elastin. The emphysematous changesspecific to this model discussed in this example can also be analyzed todetermine the effect of the antisense oligonucleotide.

Example 9 Mouse Model of Elastase Induced Emphysema

Elastase is an essential mediator in lung damage and inflammationrelease by neutrophils recruited following smoke-induced damage. A ratmodel of emphysema has been developed to analyze the process of elastasemediated lung damage, and possible therapeutic interventions to prevent,ameliorate, and/or treat the pathologies associated with such damage andresulting disease (Kuraki et al., 2002. Am J Respir Crit Care Med,166:496-500, incorporated herein by reference). Intratrachealapplication of elastase induced emphysematous changes in all lobes ofthe lung including severe lung hemorrhage as demonstrated by increasedhemoglobin in BALF; neutrophil accumulation in BALF; inhibition ofhyperinflation and degradation of elastic recoil. Histopathologicalchanges included elastase-induced airspace enlargement and breakdown ofalveoli. These changes are similar to those observed in human emphysema.

In the model, rats are treated with human sputum elastase (SE563,Elastin Products, Owensville, Mo.) without further purification. Ratsare treated with a sufficient dose of elastase, about 200 to 400 units,by intratracheal administration using a microsprayer. Alternatively,intratracheal administration can be performed as described above in themouse models. After sufficient time to allow for damage to occur, abouteight weeks, functional and morphological changes are analyzed. Asimilar model can be performed using mice with a lowered dose ofelastase relative to weight and/or lung area (e.g., 0.05 U of porcinepancreatic elastase/g body weight).

Administration of oligonucleotide can be performed prior to, concurrentwith, and/or after administration of elastase to provide a prophylacticor therapeutic model. ISIS 231894 is 100% complimentary to both mouseand rat IL 4R-α. Dose ranges are determined by the time ofoligonucleotide administration relative to elastase administration withlower doses (e.g., 1-100 ug/kg) required for prevention of lung damage.Higher doses (e.g., 100-1000 ug/kg) are required for treatment after, oralternating with, elastase administration. Positive control (e.g.,elastase treatment, no oligonucleotide administration) and negativecontrol (e.g., no elastase, with or without oligonucleotide treatment)animals are also analyzed.

Endpoints for analysis include those discussed in the asthma modelsabove. Functional endpoints include AHR, resistance and compliance.Morphological changes include BAL cell, cytokine levels, and mucusaccumulation. The emphysematous changes specific to this model discussedin this example can also be analyzed to determine the effect of theantisense oligonucleotide.

1-14. (canceled)
 15. A method for delaying or forestalling the onset ordevelopment of, ameliorating, or of treating pulmonary inflammation orairway hyperresponsiveness comprising administration of an oligomericcompound 16 to 35 nucleobases in length comprising at least 16contiguous nucleobases of the nucleobase sequence set forth in SEQ IDNO:280 to an individual in need of such intervention, thereby delayingor forestalling the onset or development of, ameliorating, or treatingpulmonary inflammation or airway hyperresponsiveness.
 16. The method ofclaim 15 wherein administration comprises topical administration to arespiratory tract of an animal.
 17. (canceled)
 18. The method of claim15 wherein administration comprises aerosol administration.
 19. Themethod of claim 15 wherein the compound is a single stranded compound.20. The method of claim 15 wherein the compound has at least onemodified internucleoside linkage, sugar moiety, or nucleobase.
 21. Themethod of claim 15 wherein the compound comprises a chimericoligonucleotide.
 22. The method of claim 20 wherein the modifiedinternucleoside linkage comprises a phosphorothioate linkage.
 23. Themethod of claim 20 wherein the modified sugar moiety comprises a2′-O-(2-methoxyethyl) (2′-MOE) modification.
 24. The method of claim 20wherein the modified nucleobase comprises 5-methylcytosine.
 25. A methodfor delaying or forestalling the onset or development of, ameliorating,or treating pulmonary inflammation or airway hyperresponsivenesscomprising administration of a compound comprising a modifiedoligonucleotide consisting of 20 linked nucleosides and having anucleobase sequence of SEQ ID NO: 280, wherein the modifiedoligonucleotide comprises: a gap segment consisting of ten linkeddeoxynucleosides; a 5′ wing segment consisting of five linkednucleosides; a 3′ wing segment consisting of five linked nucleosides;wherein the gap segment is positioned between the 5′ wing segment andthe 3′ wing segment, wherein each nucleoside of each wing segment is a2′-O-methoxyethyl nucleoside, each internucleoside linkage is aphosphorothioate internucleoside linkage and each cytosine is a5-methylcytosine, to an individual in need of such intervention, therebydelaying or forestalling the onset or development of, ameliorating, ortreating pulmonary inflammation or airway hyperresponsiveness.